Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis

ABSTRACT

Peptido and peptidomimetic compounds of the formula:                    
     wherein the formula variables are as defined in the disclosure, advantageously inhibit or block the biological activity of the picornaviral 3C protease. These compounds, as well as pharmaceutical compositions containing these compounds, are useful for treating patients or hosts infected with one or more picornaviruses, such as RVP. Intermediates and synthetic methods for preparing such compounds are also provided.

This application claims benefit of U.S. Provisional Application No.60/098,358, filed Aug. 28, 1998, and U.S. Provisional Application No.60/083,828, filed Apr. 30, 1998, which is a continuation ofPCT/US99/00260 filed Jan. 5, 1999.

FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION

The invention pertains to peptide-like and peptidomimetic compounds thatadvantageously inhibit the enzymatic activity of picornaviral 3Cproteases, especially rhinovirus 3C proteases (RVPs), and that retardviral growth in cell culture. The invention also relates to the use ofsuch compounds in pharmaceutical compositions and therapeutic treatmentsfor rhinoviral infections. The invention further relates to processesfor synthesizing such compounds and compounds useful in such syntheses.

BACKGROUND OF THE INVENTION

The picornaviruses are a family of tiny non-enveloped positive-strandedRNA-containing viruses that infect humans and other animals. Theseviruses include the human rhinoviruses, human polioviruses, humancoxsackieviruses, human echoviruses, human and bovine enteroviruses,encephalomyocarditis viruses, meningitis virus, foot and mouth viruses,hepatitis A virus, and others. The human rhinoviruses are a major causeof the common cold. To date, there are no effective therapies on themarket that cure the common cold, only treatments that relieve thesymptoms.

Picornaviral infections may be treated by inhibiting the proteolytic 3Cenzymes. These enzymes are required for the natural maturation of thepicornaviruses. They are responsible for the autocatalytic cleavage ofthe genomic, large polyprotein into the essential viral proteins.Members of the 3C protease family are cysteine proteases, where thesulfhydryl group most often cleaves the glutamine-glycine amide bond.Inhibition of 3C proteases is believed to block proteolytic cleavage ofthe polyprotein, which in turn can retard the maturation and replicationof the viruses by interfering with viral particle production. Therefore,inhibiting the processing of this cysteine protease with selective smallmolecules that are specifically recognized should represent an importantand useful approach to treat and cure viral infections of this natureand, in particular, the common cold.

Some small-molecule inhibitors of the enzymatic activity of picornaviral3C proteases (i.e., antipicornaviral compounds) have been recentlydiscovered. See, for example: U.S. patent application Ser. No.08/850,398, filed May 2, 1997, now U.S. Pat. No. 5,856,530 by Webber etal.; U.S. patent application Ser. No. 08/991,282, filed Dec. 16, 1997,U.S. Pat. No. 6,020,371 by Dragovich et al.; and U.S. patent applicationSer. No. 08/991,739, filed Dec. 16, 1997, U.S. Pat. No. 5,962,487 byWebber et al. These U.S. patent applications, the disclosures of whichare incorporated herein by reference, describe certain antipicornaviralcompounds. There is still a desire to discover small-molecule compoundsthat are especially potent antipicornaviral agents.

SUMMARY OF THE INVENTION

Thus, an object of this invention is to discover small-moleculecompounds that are especially potent antipicornaviral agents. A furtherobject of the invention is to provide intermediates useful for thesynthesis of said protease-inhibiting compounds and synthetic methodsuseful for such syntheses. A yet further object of the invention is toachieve pharmaceutical compositions that are highly effective fortreating maladies mediated by inhibition of picornaviral 3C proteases,such as the common cold.

Such objects have been attained through the discovery of compounds ofthe invention, which are picornaviral 3C protease inhibitors displayingparticularly strong antiviral activity. It has surprisingly beendiscovered that peptido and peptidomimetic compounds containing afive-membered heterocyclic group have highrhinoviral-protease-inhibiting activity. It has further beensurprisingly found that the rhinoviral-protease-inhibiting activity ofpeptido and peptidomimetic compounds may be significantly enhanced byreplacing a glutamine-like moiety found in some knownrhinoviral-protease-inhibiting compounds with a side-chain comprising agamma- or delta-lactam.

The inhibitors of the present invention are of the following generalformula (I):

wherein:

Y is —N(R_(y))—, —C(R_(y))(R_(y))—, or —O—, where each R_(y) isindependently —H or lower alkyl;

R₁ is —H, —F, -alkyl, —OH, —SH, or an O-alkyl group;

R₂ and R₃ are each independently H;

 or

 where n is an integer from 0 to 5, A₁ is CH or N, A₂ and each A₃ areindependently selected from C(R₄₁)(R₄₁), NR₄₁), S, S(O), S(O)₂, and O,and A₄ is NH or NR₄₁, where each R₄₁ is independently H or lower alkyl,provided that no more than two heteroatoms occur consecutively in theabove-depicted ring formed by A₁, A₂, (A₃)_(n), A₄ and C═O, and at leastone of R₂ and R₃ is

R₅ and R₆ are each independently H, F, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, or a heteroaryl group;

R₇ and R₈ are each independently H, an alkyl group, a cycloalkyl group,a heterocycloalkyl group, an aryl group, a heteroaryl group, —OR₁₇,—SR₁₇, —NR₁₇R₁₈, —NR₁₉NR₁₇R₁₈, or —NR₁₇OR₁₈, where R₁₇, R₁₈, and R₁₉ areeach independently H, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an aryl group, a heteroaryl group, or an acylgroup, provided that at least one of R₇ and R₈ is an alkyl group, acycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroarylgroup, —OR₁₇, —SR₁₇, —NR₁₇R₁₈, —NR₁₉NR₁₇R₁₈, or —NR₁₇OR₁₈;

R₉ is a suitable organic moiety; and

Z and Z₁ are each independently H, F, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, a heteroaryl group,—C(O)R₂₁, —CO₂R₂₁, —CN, —C(O)NR₂₁R₂₂, —C(O)NR₂₁OR₂₂, —C(S)R₂₁,—C(S)NR₂₁R₂₂, —NO₂, —SOR₂₁, —SO₂R₂₁, —SO₂NR₂₁R₂₂, —SO(NR₂₁)(OR₂₂),—SONR₂₁, —SO₃R₂₁, —PO(OR₂₁)₂, —PO(R₂₁)(R₂₂), —PO(NR₂₁R₂₂)(OR₂₃),—PO(NR₂₁R₂₂)(NR₂₃R₂₄), —C(O)NR₂₁NR₂₂R₂₃, or —C(S)NR₂₁NR₂₂R₂₃, where R₂₁,R₂₂, R₂₃, and R₂₄ are each independently H, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, a heteroaryl group, anacyl group, or a thioacyl group, or where any two of R₂₁, R₂₂, R₂₃, andR₂₄, together with the atom(s) to which they are bonded, form aheterocycloalkyl group, provided that Z and Z₁ are not both H;

or Z₁ and R₁, together with the atoms to which they are bonded, form acycloalkyl or heterocycloalkyl group, where Z₁ and R₁ are as definedabove except for moieties that cannot form the cycloalkyl orheterocycloalkyl group;

or Z and Z₁, together with the atoms to which they are bonded, form acycloalkyl or heterocycloalkyl group, where Z and Z₁ are as definedabove except for moieties that cannot form the cycloalkyl orheterocycloalkyl group.

The invention also pertains to prodrugs, pharmaceutically acceptablesalts, pharmaceutically active metabolites, and pharmaceuticallyacceptable solvates of compounds of the formula I.

In preferred embodiments of the compounds of the formula I, R₂ and R₃are each independently H;

where n is an integer from 0 to 5, each R₄₁ is independently H or loweralkyl, and the stereochemistry at the carbon denoted with an asteriskmay be R or S; provided that at least one of R₂ and R₃ is

Preferably, R₉ is a five-membered heterocycle having one to threeheteroatoms selected from O, N, and S. Alternatively, R₉ is

where R₂ is

In other preferred embodiments, the variables of formula I are asfollows. Z and Z₁ are each independently selected from H, F, loweralkyl, —CO₂R₂₁, and —C(O)NR₂₁R₂₂, provided that Z and Z₁ are not both H,where R₂₁ and R₂₂ are each independently H, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, a heteroaryl group, anacyl group, or a thioacyl group, or R₂₁ and R₂₂, together with theatom(s) to which they are bonded, form a heterocycloalkyl group. Atleast one of R₂ or R₃ is

and the other is H. R₅ and R₆ are each independently selected from H, F,an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an arylgroup, and a heteroaryl group, more preferably one of R₅ and R₆ is H andthe other is alkyl or aryl (e.g., unsubstituted or substitutedphenylmethyl). R₇ and R₈ are each independently H, an alkyl group, acycloalkyl group, a heterocycloalkyl group, an aryl group, or aheteroaryl group; and more preferably one of R₇ and R₈ is H and theother is alkyl (e.g., 2-propyl, 2-methyl-2-propyl, or 2-methyl-1-propyl)or arylmethyl (e.g., unsubstituted or substituted phenylmethyl ornaphthylmethyl). R₉ is a five-membered heterocycle having from one tothree heteroatoms selected from O, N, and S, more preferably afive-membered heterocycle having at least one nitrogen heteroatom and atleast one oxygen heteroatom (e.g., unsubstituted or substituted1,2-oxazolyl (i.e., isoxazolyl), 1,3-oxazolyl (i.e., oxazolyl), oroxadiazolyl (1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, or1,2,5-oxadiazolyl). When R₉ is oxadiazolyl, unsubstituted andmonomethyl-substituted 1,2,4-oxadiazolyl are preferred. In especiallypreferred embodiments, R₉ is 3-isoxazolyl or 5-isoxazolyl, eitherunsubstituted or substituted with one or two methyl groups and/orhalogens, with chlorine and fluorine being preferred halogensubstituents.

In a preferred embodiment, the compounds, prodrugs, pharmaceuticallyacceptable salts, pharmaceutically active metabolites, and solvates havean antipicornaviral activity with an EC₅₀ less than or equal to 100 μMin the H1-HeLa cell culture assay, and more preferably an antirhinoviralactivity with an EC₅₀ less than or equal to 10 μM in the H1-HeLa cellculture.

In another aspect, the invention is directed to intermediates of formulaII, preferably of the formula II′, which are useful in the synthesis ofcertain compounds:

wherein:

p is an integer of from 0 to 5;

A₁₁ is CH or N, A₁₂ and each A₁₃ are independently selected fromC(R₆₁)(R₆₁), N(R₆₁), S, S(O), S(O)₂, and O, and A₁₄ is NH or NR₆₁, whereeach R₆₁ is independently H, alkyl, acyl, or aryl, provided that no morethan two heteroatoms occur consecutively in the above-depicted ring informula II formed by A₁₁, A₁₂, (A₁₃)_(n), A₁₄ and C═O;

each R₁₄₁ is independently H or lower alkyl;

R₅, is H, alkyl, acyl, or aryl;

R₅₂, R₅₃, and R₅₄ are each independently selected from H, hydroxyl,alkyl, acyl, and aryl; or any two of R₅₂, R₅₃, and R₅₄ together form ═Oor ═C(R₅₇)(R₅₈), where R₅₇ and R₅₈ are each independently selected fromH, alkyl, CO₂(C₁-C₆)alkyl, C(O)N(C₁-C₆)alkyl, and CO₂(aryl); and

R₅₅ and R₅₆ are each independently H or a suitable protecting group fornitrogen.

The invention is also directed to pharmaceutically acceptable salts ofthe compounds of formulae II and II′.

The invention also relates to pharmaceutical compositions containing atherapeutically effective amount of at least one compound of the formulaI, or a prodrug, pharmaceutically acceptable salt, pharmaceuticallyactive metabolite, or solvate thereof. Additionally, the inventionrelates to methods of inhibiting picornaviral 3C protease byadministering a therapeutically effective amount of at least onecompound of the formula I, or a prodrug, pharmaceutically acceptablesalt, pharmaceutically active metabolite, or solvate thereof.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

The present invention relates to compounds of the formula I:

wherein Y, R₁, R₂, R₃, R₅, R₆, R₇, R₈, R₉, Z, and Z₁ are as definedabove, and to pharmaceutically acceptable salts, prodrugs, activemetabolites, and solvates thereof. Preferably, such compounds,pharmaceutically acceptable salts, prodrugs, active metabolites, andsolvates have antipicornaviral activity, more preferably antirhinoviralactivity, corresponding to an EC₅₀ less than or equal to 100 μM in theH1-HeLa cell culture assay, more preferably corresponding to an EC₅₀less than or equal to 10 μM in the H1-HeLa cell culture assay.

The present invention additionally relates to preferred compounds of theformulas I-A, I-B, and I-C:

wherein R_(y) (in formula I-A) is H or lower alkyl, and R₁, R₂, R₃, R₅,R_(6,) R₇, R₈, R₉, Z, and Z₁ are as defined above, and topharmaceutically acceptable salts, prodrugs, active metabolites, andsolvates thereof.

The inventive compounds of formulas I-A, which are referred to herein as“peptide-like” compounds, I-B, which are referred to herein as“ketomethylene-type” compounds, and I-C, which are referred to herein as“depsipeptide” compounds, differ in their backbones, which may affectthe specific biodistribution or other physical properties; nonethelesseach possesses a strong rhinoviral-protease-inhibiting activity.

In preferred embodiments of compounds of formulas I-A, I-B, and I-Cabove:

R₁ is H, F, or an alkyl group;

R_(y) (in formula I-A) is H or methyl;

R₃, R₅, and R₈ are each H;

R₂ is selected from one of the following moieties:

R₆ is an alkyl group, which has as a preferred optional substituent anaryl group;

R₇ is an alkyl group, a cycloalkyl group, a heterocycloalkyl group, anaryl group, or a heteroaryl group;

R₉ is a five-membered heterocycle having from one to three heteroatomsselected from O, N, and S, preferably where at least one of theheteroatoms is nitrogen, that is unsubstituted or substituted, where theoptional substituents are preferably halogen or lower alkyl, and morepreferably mono-chloro or -fluoro or a methyl group; and

Z and Z₁ are each independently H, F, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, a heteroaryl group,—C(O)R₂₁, —CO₂R₂₁, —CN, —C(O)NR₂₁R₂₂, —C(O)NR₂₁OR₂₂, —C(S)R₂₁,—C(S)NR₂₁R₂₂, —NO₂, —SOR₂₁, —SO₂R₂₁, —SO₂NR₂₁R₂₂, —SO(NR₂₁)(OR₂₂),—SONR₂₁, —SO₃R₂₁, —PO(OR₂₁)₂, —PO(R₂₁)(R₂₂), —PO(NR₂₁R₂₂)(OR₂₃),—PO(NR₂₁R₂₂)(NR₂₃R₂₄), —C(O)NR₂₁NR₂₂R₂₃, or —C(S)NR₂₁NR₂₂R₂₃, where Zand Z₁ are not both H, and where R₂₁, R₂₂, R₂₃, and R₂₄ are eachindependently H, an alkyl group, a cycloalkyl group, a heterocycloalkylgroup, an aryl group, a heteroaryl group, an acyl group, or a thioacylgroup, or where any two of R₂₁, R₂₂, R₂₃, and R₂₄, together with theatom(s) to which they are bonded, form a heterocycloalkyl group,

or Z and Z₁ (both as defined above), together with the atoms to whichthey are attached, form a heterocycloalkyl group.

In preferred embodiments, the compounds of the invention are of theformulae I-A′, I-B′, and I-C′:

wherein:

R₁, Z, and Z₁ are as defined above;

n is 1 or 2;

R_(y) (in formula I-A′) is H or lower alkyl;

R₆ is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R₇ is alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —OR₁₇,—SR₁₇, —NR₁₇R₁₈, —NR₁₉NR₁₇R₁₈, or —NR₁₇OR₁₈, where R₁₇, R₁₈, and R₁₉ areeach independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, or acyl;

R₉ is a five-membered heterocycle having one to three heteroatomsselected from O, N, and S, that is unsubstituted or substituted, wherethe optional substituents are preferably one or two lower alkyl groupsand/or halogens; and

R₂₀ is H.

The invention also relates to prodrugs, pharmaceutically acceptablesalts, pharmaceutically active metabolites, and solvate of suchcompounds.

In preferred embodiments, the RVP-inhibiting agents of the invention arecompounds of any of the stereospecific formulas I-A″, I-B″, and I-C″:

wherein R_(y), R₁, R₂, R₆, R₇, R₉, Z, and Z₁ are as defined above, andpharmaceutically acceptable salts, prodrugs, active metabolites, andsolvates thereof.

In preferred embodiments of compounds of the formula I-A″, I-B″, orI-C″:

R₁ is H, F, or methyl;

R_(y) (in formula I-A′) is H or methyl;

R₂ is selected from one of the following moieties:

R₆ is arylmethyl or arylthiomethyl, where aryl is preferably anoptionally substituted phenyl group;

R₇ is an alkyl group, more preferably selected from 2-propyl,2-methyl-2-propyl, 2-methyl-1-propyl, and arylmethyl, where the arylgroup is preferably phenyl or naphthyl;

R₉ is isoxazolyl, oxazolyl, or oxadiazolyl, optionally substituted withone or two lower alkyl groups and/or halogens; and

Z is H, and Z₁ is —CO₂R₂₁, —CN, or —C(O)NR₂₁R₂₂, where R₂₁ and R₂₂ areas defined above, or Z and Z₁ together form a cyclic ester or amide.

Even more preferably, the RVP-inhibiting agents of the invention arecompounds of any of the formulas I-A′″, I-B′″, and I-C′″:

wherein n, R_(y), R₁, R₂₀, R₆, R₇, R₉, Z, and Z₁ are as defined above,and pharmaceutically acceptable salts, prodrugs, active metabolites, andsolvates thereof.

In preferred compounds of the formula (I-A′″), (I-B′″), or (I-C′″):

R₁ is H, F, or methyl;

R_(y) (in formula I-A′) is H or methyl;

R₂₀ is hydrogen;

R₆ is arylmethyl or arylthiomethyl, where aryl is preferably phenylunsubstituted or substituted with halogen, lower alkyl, and/or loweralkoxy;

R₇ is an alkyl group, and more preferably is selected from 2-propyl,2-methyl-2-propyl, 2-methyl-1-propyl, and arylmethyl, where the arylgroup is preferably phenyl or naphthyl;

R₉ is isoxazolyl, oxazolyl, or oxadiazolyl, each optionally substitutedwith one or two lower alkyl groups and/or halogens; and

Z is H, and Z₁ is —CO₂R₂₁, —CN, or —C(O)NR₂₁R₂₂, where R₂₁ and R₂₂ areas defined above, or Z and Z₁ together form a cyclic ester or amide.

In especially preferred compounds of the invention of the genericformula I (and subgeneric formulae), R₁ is H or F.

In another aspect, the invention is directed to intermediate compoundsof the formulas II and II′:

wherein the variables (p, A₁₁, A₁₂, A₁₃, A₁₄, R₅₁, R₅₂, R₅₃, R₅₄, R₅₅,R₅₆, and R₁₄₁) are as defined above. These compounds are useful forsynthesizing pharmaceutically useful compounds of the formula I.

Preferred R₅₅ and R₅₆ groups are H and suitable protecting groups fornitrogen, for example, BOC (t-butyloxycarbonyl), CBZ(benzyloxycarbonyl), FMOC (fluorene-9-methyloxycarbonyl), otheralkyloxycarbonyls (e.g. methyloxycarbonyl), and trityl(triphenylmethyl). Other suitable nitrogen-protecting groups may bereadily selected by artisans (see, e.g., Greene and Wutz, ProtectingGroups in Chemical Synthesis (2^(nd) ed.), John Wiley & Sons, NY(1991)). Preferred groups for R₅₂, R₅₃, and R₅₄ are H, alkoxy, hydroxy,and carbonyl.

Preferred formula-II compounds include the following, where P_(N) is asuitable protecting group for nitrogen and q is 1 or 2:

Other preferred intermediates include the following compounds, where BOCis t-butyloxycarbonyl:

Of these, the preferred stereoisomers are:

Especially preferred intermediates include the following compounds:

In accordance with a convention used in the art,

is used in structural formulas herein to depict the bond that is thepoint of attachment of the moiety or substituent to the core or backbonestructure.

Where chiral carbons are included in chemical structures, unless aparticular orientation is depicted, both stereoisomeric forms areintended to be encompassed.

As used herein, the term “alkyl group” is intended to mean a straight-or branched-chain monovalent radical of saturated and/or unsaturatedcarbon atoms and hydrogen atoms, such as methyl (Me), ethyl (Et),propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, pentenyl, butenyl,propenyl, ethynyl, butynyl, propynyl, pentynyl, hexynyl, and the like,which may be unsubstituted (i.e., containing only carbon and hydrogen)or substituted by one or more suitable substituents as defined below(e.g., one or more halogens, such as F, Cl, Br, or I, with F and Clbeing preferred). A “lower alkyl group” is intended to mean an alkylgroup having from 1 to 4 carbon atoms in its chain.

A “cycloalkyl group” is intended to mean a non-aromatic monovalentmonocyclic, bicyclic, or tricyclic radical containing 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 carbon ring atoms, each of which may besaturated or unsaturated, and which may be unsubstituted or substitutedby one or more suitable substituents as defined below, and to which maybe fused one or more heterocycloalkyl groups, aryl groups, or heteroarylgroups, which themselves may be unsubstituted or substituted by one ormore substituents. Illustrative examples of cycloalkyl groups includethe following moieties:

A “heterocycloalkyl group” is intended to mean a non-aromatic monovalentmonocyclic, bicyclic, or tricyclic radical, which is saturated orunsaturated, containing 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 ring atoms, which includes 1, 2, 3, 4, or 5 heteroatomsselected from nitrogen, oxygen, and sulfur, where the radical isunsubstituted or substituted by one or more suitable substituents asdefined below, and to which may be fused one or more cycloalkyl groups,aryl groups, or heteroaryl groups, which themselves may be unsubstitutedor substituted by one or more suitable substituents. Illustrativeexamples of heterocycloalkyl groups include the following moieties:

An “aryl group” is intended to mean an aromatic monovalent monocyclic,bicyclic, or tricyclic radical containing 6, 10, 14, or 18 carbon ringatoms, which may be unsubstituted or substituted by one or more suitablesubstituents as defined below, and to which may be fused one or morecycloalkyl groups, heterocycloalkyl groups, or heteroaryl groups, whichthemselves may be unsubstituted or substituted by one or more suitablesubstituents. Illustrative examples of aryl groups include the followingmoieties:

A “heteroaryl group” is intended to mean an aromatic monovalentmonocyclic, bicyclic, or tricyclic radical containing 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, or 18 ring atoms, including 1, 2, 3, 4,or 5 heteroatoms selected from nitrogen, oxygen, and sulfur, which maybe unsubstituted or substituted by one or more suitable substituents asdefined below, and to which may be fused one or more cycloalkyl groups,heterocycloalkyl groups, or aryl groups, which themselves may beunsubstituted or substituted by one or more suitable substituents.Illustrative examples of heteroaryl groups include the followingmoieties:

A “heterocycle” is intended to mean a heteroaryl or heterocycloalkylgroup (each of which, as defined above, are optionally substituted).

An “acyl group” is intended to mean a —C(O)—R radical, where R is asubstituent as defined below.

A “thioacyl group” is intended to mean a —C(S)—R radical, where R is asubstituent as defined below.

A “sulfonyl group” is intended to mean a —SO₂R radical, where R is asubstituent as defined below.

A “hydroxy group” is intended to mean the radical —OH.

An “amino group” is intended to mean the radical —NH₂.

An “alkylamino group” is intended to mean the radical —NHR_(a), whereR_(a) is an alkyl group.

A “dialkylamino group” is intended to mean the radical —NR_(a)R_(b),where R_(a) and R_(b) are each independently an alkyl group.

An “alkoxy group” is intended to mean the radical —OR_(a), where R_(a)is an alkyl group. Exemplary alkoxy groups include methoxy, ethoxy,propoxy, and the like.

An “alkoxycarbonyl group” is intended to mean the radical —C(O)OR_(a),where R_(a) is an alkyl group.

An “alkylsulfonyl group” is intended to mean the radical —SO₂R_(a),where R_(a) is an alkyl group.

An “alkylaminocarbonyl group” is intended to mean the radical—C(O)NHR_(a), where R_(a) is an alkyl group.

A “dialkylaminocarbonyl group” is intended to mean the radical—C(O)NR_(a)R_(b), where R_(a) and R_(b) are each independently an alkylgroup.

A “mercapto group” is intended to mean the radical —SH.

An “alkylthio group” is intended to mean the radical —SR_(a), whereR_(a) is an alkyl group.

A “carboxy group” is intended to mean the radical —C(O)OH.

A “carbamoyl group” is intended to mean the radical —C(O)NH₂.

An “aryloxy group” is intended to mean the radical —OR_(c), where R_(c)is an aryl group.

A “heteroaryloxy group” is intended to mean the radical —OR_(d), whereR_(d) is a heteroaryl group.

An “arylthio group” is intended to mean the radical —SR_(c), where R_(c)is an aryl group.

A “heteroarylthio group” is intended to mean the radical —SR_(d), whereR_(d) is a heteroaryl group.

The term “suitable organic moiety” is intended to mean any organicmoiety recognizable, such as by routine testing, to those skilled in theart as not adversely affecting the inhibitory activity of the inventivecompounds. Illustrative examples of suitable organic moieties include,but are not limited to, hydroxy groups, alkyl groups, oxo groups,cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroarylgroups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups,alkoxy groups, carboxy groups, amino groups, alkylamino groups,dialkylamino groups, carbamoyl groups, arylthio groups, heteroarylthiogroups, and the like.

The term “substituent” or “suitable substituent” is intended to mean anysuitable substituent that may be recognized or selected, such as throughroutine testing, by those skilled in the art. Illustrative examples ofsuitable substituents include hydroxy groups, halogens, oxo groups,alkyl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthiogroups, alkoxy groups, cycloalkyl groups, heterocycloalkyl groups, arylgroups, heteroaryl groups, carboxy groups, amino groups, alkylaminogroups, dialkylamino groups, carbamoyl groups, aryloxy groups,heteroaryloxy groups, arylthio groups, heteroarylthio groups, and thelike.

The term “optionally substituted” is intended to expressly indicate thatthe specified group is unsubstituted or substituted by one or moresuitable substituents, unless the optional substituents are expresslyspecified, in which case the term indicates that the group isunsubstituted or substituted with the specified substituents. As definedabove, various groups may be unsubstituted or substituted (i.e., theyare optionally substituted) unless indicated otherwise herein (e.g., byindicating that the specified group is unsubstituted).

A “prodrug” is intended to mean a compound that is converted underphysiological conditions or by solvolysis or metabolically to aspecified compound that is pharmaceutically active.

A “pharmaceutically active metabolite” is intended to mean apharmacologically active product produced through metabolism in the bodyof a specified compound.

A “solvate” is intended to mean a pharmaceutically acceptable solvateform of a specified compound that retains the biological effectivenessof such compound. Examples of solvates include compounds of theinvention in combination with water, isopropanol, ethanol, methanol,DMSO, ethyl acetate, acetic acid, or ethanolamine.

A “pharmaceutically acceptable salt” is intended to mean a salt thatretains the biological effectiveness of the free acids and bases of thespecified compound and that is not biologically or otherwiseundesirable. Examples of pharmaceutically acceptable salts includesulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates,citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates,methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, and mandelates.

If an inventive compound is a base, a desired salt may be prepared byany suitable method known to the art, including treatment of the freebase with an inorganic acid, such as hydrochloric acid; hydrobromicacid; sulfuric acid; nitric acid; phosphoric acid; and the like, or withan organic acid, such as acetic acid; maleic acid; succinic acid;mandelic acid; fumaric acid; malonic acid; pyruvic acid; oxalic acid;glycolic acid; salicylic acid; pyranosidyl acid, such as glucuronic acidor galacturonic acid; alpha-hydroxy acid, such as citric acid ortartaric acid; amino acid, such as aspartic acid or glutamic acid;aromatic acid, such as benzoic acid or cinnamic acid; sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid; or the like.

If an inventive compound is an acid, a desired salt may be prepared byany suitable method known to the art, including treatment of the freeacid with an inorganic or organic base, such as an amine (primary,secondary, or tertiary); an alkali metal or alkaline earth metalhydroxide; or the like. Illustrative examples of suitable salts includeorganic salts derived from amino acids such as glycine and arginine;ammonia; primary, secondary, and tertiary amines; and cyclic amines,such as piperidine, morpholine, and piperazine; as well as inorganicsalts derived from sodium, calcium, potassium, magnesium, manganese,iron, copper, zinc, aluminum, and lithium.

In the case of compounds, salts, or solvates that are solids, it isunderstood by those skilled in the art that the inventive compounds,salts, and solvates may exist in different crystal forms, all of whichare intended to be within the scope of the present invention andspecified formulas.

The inventive compounds may exist as single stereoisomers, racemates,and/or mixtures of enantiomers and/or diastereomers. All such singlestereoisomers, racemates, and mixtures thereof are intended to be withinthe broad scope of the present invention. Preferably, however, theinventive compounds are used in optically pure form.

As generally understood by those skilled in the art, an optically purecompound is one that is enantiomerically pure. As used herein, the term“optically pure” is intended to mean a compound comprising at least asufficient activity. Preferably, an optically amount of a singleenantiomer to yield a compound having the desired pharmacological purecompound of the invention comprises at least 90% of a single isomer (80%enantiomeric excess), more preferably at least 95% (90% e.e.), even morepreferably at least 97.5% (95% e.e.), and most preferably at least 99%(98% e.e.).

Preferably in the compounds of the formula I (or of any of thesubgeneric formula), R₁ is H or F.

In the compounds of formula I, preferably R₉ is an unsubstituted orsubstituted isoxazolyl group, where the optional substituents arepreferably one or two methyl groups and/or halogens.

Especially preferred embodiments of the invention are described below inreference to the following formula I-A″:

Preferred compounds of the present invention include peptido(peptide-like) Compounds (A-1)-(A-8) of the formula I-A″ above, whereinR₁ is H, Z is H, R_(y) is H, and R₂, R₆, R₇, Z₁, and R₉ are asrespectively defined below:

(A-1) R₂ is CH₂CH₂C(O)NH₂, R₆ is CH₂Ph, R₇ is CH₂CH(CH₃)₂, Z₁ isCO₂CH₂CH₃, and R₉ is

(A-2) R₂ is CH₂CH₂C(O)NH₂, R₆ is CH₂Ph, R₇ is CH₂CH(CH₃)₂, Z₁ isCO₂CH₂CH₃, and R₉ is

(A-3) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is C(CH₃)₃, Z₁ is CO₂CH₂CH₃, and R₉ is

(A-4) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is C(CH₃)₃, Z₁ is CO₂CH₂CH₃, and R₉ is

(A-5) R₂ is

R₆ is

R₇ is CH(CH₃)₂, Z₁ is CO₂CH₂CH₃, and R₉ is

(A-6) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is CH(CH₃)₂, Z₁ is CO₂CH₂CH₃, and R₉ is

(A-7) R₂ is

R₆ is

R₇ is C(CH₃)₃, Z₁ is CO₂CH₂CH₃, and R₉ is

(A-8) R₂ is

R₆ is

R₇ is CH(CH₃)₂, Z₁ is CO₂CH₂CH₃, and R₉ is

Preferred peptide-like compounds of the formula I-A″″ further includeCompounds (A-9)-(A-13) below, wherein R₁ is H, Z is H, Z₁ is CO₂CH₂CH₃,R_(y) is CH₃, and R₂, R₆, R₇, and R₉ are as respectively defined below:

(A-9) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is

and R₉ is

(A-10) R₂ is CH₂CH₂C(O)NH₂, R₆ is CH₂Ph, R₇ is CH₂CH(CH₃)₂, and R₉ is

(A-11) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is

and R₉ is

(A-12) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is CH₂CH(CH₃)₂, and R₉ is

(A-13) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is

and R₉ is

Other preferred peptide-like compounds include the following:

Preferred ketomethylene-type Compounds (B-1)-(B-4) of the invention aredescribed below in reference to the following formula I-B″:

(B-1) R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is CH(CH₃)₂, Z is H, Z₁ is CO₂CH₂CH₃, and R₉ is

(B-2) R₂ is

R₆ is

R₇ is CH(CH₃)₂, Z is H, Z₁ is CO₂CH₂CH₃, and R₉ is

(B-3) R₂ is

R₆ is

R₇ is CH(CH₃)₂, Z and Z₁ together are

where the carbonyl group is cis to the hydrogen corresponding to R₁ informula I, and R₉ is

(B-4) R₂ is

R₆ is

R₇ is CH(CH₃)₂, Z is H, Z₁ is CO₂CH₂CH₃, and R₉ is

Preferred depsipeptide-type Compounds (C-1) and (C-2) of the inventionare described below in reference to the following formula I-C″, where R₁is H:

(C-1) Z is H, R₂ is CH₂CH₂C(O)NH₂, R₆ is

R₇ is CH(CH₃)₂, Z₁ is CO₂CH₂CH₃, and R₉ is

(C-2) Z is H, R₂ is

R₆ is

R₇ is CH(CH₃)₂, Z₁ is CO₂CH₂CH₃, and R₉ is

Additional compounds may be prepared in reference to formula I byselecting the variables from the following substituents:

R_(y)=H or CH₃;

R₁=H or CH₃;

 or phenylmethyl (i.e., benzyl), where the aryl group is optionallysubstituted with one, two, or three substituents each independentlyselected from halogens, methoxy and methyl;

R₇=2-methyl-1-propyl, 2-propyl, 2-methyl-2-propyl, benzyl, or

 and

The present invention is also directed to a method of inhibitingpicornaviral 3C protease activity, comprising contacting the proteasewith an effective amount of a compound of formula I, or apharmaceutically acceptable salt, prodrug, pharmaceutically activemetabolite, or solvate thereof. For example, picornaviral 3C proteaseactivity may be inhibited in mammalian tissue by administering acompound of formula I or a pharmaceutically acceptable salt, prodrug,pharmaceutically active metabolite, or solvate thereof. More preferably,the present method is directed at inhibiting rhinoviral proteaseactivity.

“Treating” or “treatment” is intended to mean at least the mitigation ofa disease condition in a mammal, such as a human, that is alleviated bythe inhibition of the activity of one or more picornaviral 3C proteases,such as human rhinoviruses, human poliovirus, human coxsackieviruses,encephalomyocarditis viruses, meningitis virus, and hepatitis A virus,and includes: (a) prophylactic treatment in a mammal, particularly whenthe mammal is found to be predisposed to having the disease conditionbut not yet diagnosed as having it; (b) inhibiting the diseasecondition; and/or (c) alleviating, in whole or in part, the diseasecondition.

The activity of the inventive compounds as inhibitors of picornaviral 3Cprotease activity may be measured by any of the suitable methods knownto those skilled in the art, including in vivo and in vitro assays. Anexample of a suitable assay for activity measurements is the antiviralH1-HeLa cell culture assay described herein.

Administration of the compounds of the formula I and theirpharmaceutically acceptable prodrugs, salts, active metabolites, andsolvates may be performed according to any of the accepted modes ofadministration available to those skilled in the art. Illustrativeexamples of suitable modes of administration include oral, nasal,parenteral, topical, transdermal, and rectal. Intranasal delivery isespecially preferred.

An inventive compound of formula I or a pharmaceutically acceptablesalt, prodrug, active metabolite, or solvate thereof may be administeredas a pharmaceutical composition in any pharmaceutical form recognizableto the skilled artisan as being suitable. Suitable pharmaceutical formsinclude solid, semisolid, liquid, or lyophilized formulations, such astablets, powders, capsules, suppositories, suspensions, liposomes, andaerosols. Pharmaceutical compositions of the invention may also includesuitable excipients, diluents, vehicles, and carriers, as well as otherpharmaceutically active agents, depending upon the intended use. Inpreferred embodiments, the inventive pharmaceutical compositions aredelivered intranasally in the form of suspensions.

Acceptable methods of preparing suitable pharmaceutical forms of thepharmaceutical compositions are known or may be routinely determined bythose skilled in the art. For example, pharmaceutical preparations maybe prepared following conventional techniques of the pharmaceuticalchemist involving steps such as mixing, granulating, and compressingwhen necessary for tablet forms, or mixing, filling, and dissolving theingredients as appropriate, to give the desired products for oral,parenteral, topical, intravaginal, intranasal, intrabronchial,intraocular, intraaural, and/or rectal administration.

Solid or liquid pharmaceutically acceptable carriers, diluents,vehicles, or excipients may be employed in the pharmaceuticalcompositions. Illustrative solid carriers include starch, lactose,calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, pectin,acacia, magnesium stearate, and stearic acid. Illustrative liquidcarriers include syrup, peanut oil, olive oil, saline solution, andwater. The carrier or diluent may include a suitable prolonged-releasematerial, such as glyceryl monostearate or glyceryl distearate, alone orwith a wax. When a liquid carrier is used, the preparation may be in theform of a syrup, elixir, emulsion, soft gelatin capsule, sterileinjectable liquid (e.g., solution), or a nonaqueous or aqueous liquidsuspension.

A dose of the pharmaceutical composition contains at least atherapeutically effective amount of the active compound (i.e., acompound of formula I or a pharmaceutically acceptable salt, prodrug,active metabolite, or solvate thereof), and preferably is made up of oneor more pharmaceutical dosage units. The selected dose may beadministered to a mammal, for example, a human patient, in need oftreatment mediated by inhibition of picornaviral 3C protease activity,by any known or suitable method of administering the dose, includingtopically, for example, as an ointment or cream; orally; rectally, forexample, as a suppository; parenterally by injection; or continuously byintravaginal, intranasal, intrabronchial, intraaural, or intraocularinfusion.

A “therapeutically effective amount” is intended to mean the amount ofan inventive compound that, when administered to a mammal in needthereof, is sufficient to effect treatment for disease conditionsalleviated by the inhibition of the activity of one or more picornaviral3C proteases, such as human rhinoviruses, human poliovirus, humancoxsackieviruses, encephalomyocarditis viruses, menigovirus, andhepatitis A virus. The amount of a given compound of the invention thatwill be therapeutically effective will vary depending upon factors suchas the particular compound, the disease condition and the severitythereof, the identity of the mammal in need thereof, which amount may beroutinely determined by artisans.

By way of illustration, a formulation for nasal delivery of theinventive compounds for treatment of rhinoviral infections can beprepared as follows, where all percentages are weight/weight and thesuspension is prepared in purified water. A formula-I compound ismicronized to a reduced particle size such that D₉₀<10 μm. A suspensionis prepared to contain a final concentration of from about 0.01% toabout 2% of the active compound, preferably about from 0.2% to 2%. Anappropriate preservative selected from those known in the art may beincluded, for example, benzalkonium chloride/EDTA, in appropriatefinal-concentration ranges, e.g., about 0.02%/0.01%. A suspending agent,such as mixture of microcrystalline cellulose (final concentration ofabout 1%-1.5%, preferably about 1.2%) and sodium carboxymethylcellulosecellulose (final concentration of about 0.1%-0.2%, preferably about0.13%) may be included. A surfactant such as polysorbate 80 may beincluded in a final concentration of about from 0.05% to 0.2%,preferably about 0.1%. A tonicity modifier such as dextrose may beincluded to give a final concentration of about from 4% to 6%,preferably about 5%. The pH of the final solution is adjusted asappropriate to a physiological range, e.g., 4-6, using non-toxic acidand/or base, such as HCl and/or NaOH.

An exemplary formulation for nasal delivery of the inventive compound ofExample 17 has the following composition, where all percentages areweight/weight and the suspension is prepared in purified water:

Active Compound (B-2) 0.2-2%   Preservative Benzalkonium 0.02%/0.01%chloride/EDTA Suspending Agent Microcrystalline cellulose/Na- 1.2%/0.13% carboxymethylcellulose Surfactant Polysorbate 80 0.1%Tonicity Modifier Dextrose   5% pH Adjustment NaOH/HCl pH 4-6

GENERAL SYNTHESES

The inventive compounds of formula (I) may be advantageously prepared bythe methods of the present invention, including the general methodsdescribed below. In each of these general methods, R₁, R₂, R₃, R₅, R₆,R₇, R₈, R₉, R_(y), Z, and Z₁ are as defined above, and R₄ is used (as ashorthand representation) to mean:

where R₇, R₈, and R₉ are as defined above.

In General Method I, useful for synthesis of peptide-like compounds offormula I-A, an amino acid A, where P₁ is an appropriate protectinggroup for nitrogen, is subjected to an amide-forming reaction with aminoalcohol (or salt thereof) B to produce amide C. Compound C is thendeprotected to give free amine (or salt thereof) D. Amine D and aminoacid E, which may incorporate either an R₄ group or a protecting groupfor nitrogen (P₂), are subjected to a bond-forming reaction generatingcompound F. Compound F is oxidized to intermediate G, which is thentransformed into unsaturated product H. If protecting groups have beenused on amino acid E, or on any R groups (R₁-R₉) and/or on R_(y) and/oron Z and/or Z₁, product H is deprotected and/or further modified toyield deprotected or modified H.

An alternative method to prepare intermediate F is described as follows.Amino acid E and amino acid (or salt thereof) I, where P₃ is anappropriate protecting group for oxygen, are subjected to a bond-formingreaction to produce intermediate J. Molecule J is deprotected to yieldfree carboxylic acid K, which is subsequently subjected to anamide-forming reaction with amino alcohol (or salt thereof) B togenerate intermediate F.

In General Method II, which is also useful for synthesizing peptide-likecompounds of formula I-A, an amino acid L, where P₁ is an appropriateprotecting group for nitrogen, is converted to a carbonyl derivative M,where “Lv” is a leaving group. Compound M is subjected to a reactionwhere Lv is replaced by R₁ to give derivative N. Derivative N is thentransformed into unsaturated product O. Unsaturated compound O isdeprotected to give free amine (or salt thereof) P, or modified at Z orZ₁ first to give O′, and then deprotected to P. Intermediate P issubsequently subjected to an amide-forming reaction with carboxylic acidK to give final product H. If protecting groups have been used on any Rgroup (R₁-R₉) and/or on R_(y) and/or on Z and/or Z₁, product H isdeprotected and/or further modified to yield deprotected or modified H.

An alternative method to prepare intermediate N is described as follows.Compound M is subjected to a reaction where “Lv” (or more particularly—C(O)—Lv), is reduced to protected amino alcohol Q. Intermediate Q issubsequently oxidized to derivative N.

In General Method III, useful for synthesis of peptide-like compounds offormula I-A, an amino acid L, where P₁ is an appropriate protectinggroup for nitrogen, is converted to a carbonyl derivative M, where “Lv”is a leaving group. Compound M is deprotected to give free amine (orsalt thereof) R, which subsequently is subjected to an amide-formingreaction with carboxylic acid K to give intermediate S. Compound S isthen either directly converted to carbonyl intermediate G, orsuccessively reduced to alcohol F first, which is oxidized to G.Compound G is subjected to a reaction to yield the unsaturated finalproduct H. If protecting groups have been used on any R groups (R₁-R₉)and/or on R_(y) and/or on Z and/or Z₁, product H is deprotected and/orfurther modified to yield deprotected or modified H.

In General Method IV, useful for synthesis of peptide-like compounds offormula I-A, free amine (or salt thereof) P, prepared from intermediateO as described in General Method II, is converted to amide T by reactionwith amino acid A, where P₁ is an appropriate protecting group fornitrogen. Compound T is further deprotected to free amine (or saltthereof) U, which is subsequently converted to H with amino acid E. Ifprotecting groups have been used on any R groups (R₁-R₉) and/or on R_(y)and/or on Z and/or Z₁, product H is deprotected and/or further modifiedto yield deprotected or modified H.

In General Method V, useful for synthesis of ketomethylene compounds offormula I-B, optically active lactone AA, where P₄ is an appropriateprotecting group for nitrogen, and R₅ and R₈ are H (which may beprepared by the method described below and by various literaturemethods, including: (a) Herold et al., J. Org. Chem. 1989, 54, 1178; (b)Bradbury et al., Tetrahedron Lett. 1989, 30, 3845; (c) Bradbury et al.,J. Med. Chem. 1990, 33, 2335; (d) Wuts et al., J. Org. Chem. 1992, 57,6696; (e) Jones et al., J. Org. Chem. 1993, 58, 2286; (f) Pégorier etal., Tetrahedron Lett. 1995, 36, 2753; and (g) Dondoni et al., J. Org.Chem. 1995, 60, 7927) is transformed by a two-step procedure (basichydrolysis and subsequent oxidation) into carboxylic acid BB. Thismaterial is not isolated, but is subjected to an amide-forming reactionwith amine (or salt thereof) P to provide final product CC. The P₄protecting group, along with any additional protecting groups that havebeen used on any R groups (R₁, R₂, R₃, R₆, and/or R₇) and/or on Z and/oron Z₁, is subsequently deprotected and/or further modified to yielddeprotected or modified CC.

Lactone AA may be prepared in optically active form by the followingGeneral Method VI (see: Herold et al., J. Org. Chem. 1989, 54, 1178;Bradbury et al., Tetrahedron Lett. 1989, 30, 3845; and Bradbury et al.,J. Med. Chem. 1990, 33, 2335). A γ,δ-unsaturated carboxylic acid DD,which incorporates R₇, is transformed into the corresponding acidchloride (not shown). This acid chloride is subjected to anamide-forming reaction with a chiral amine or a chiral oxazolidone toprovide derivative EE (in which X₁ is a chiral amine or a chiraloxazolidone). Compound EE is subsequently deprotonated, and theresulting enolate is diastereoselectively alkylated with an electrophilecorresponding to R₆ to provide compound FF, where R₅ is H. This materialis then subjected to a halolactonization reaction to providehalo-lactone GG, in which R₅ and R₈ are H and “hal” is Br or I.Halo-lactone GG is subsequently transformed into azide HH, and thismaterial is then converted into lactone AA, where P₄ is an appropriateprotecting group for nitrogen.

γ,δ-Unsaturated carboxylic acid DD may be prepared by the followingGeneral Method VII (see: Herold et al., J. Org. Chem. 1989, 54, 1178).An aldehyde II, which incorporates R₇, is coupled with vinylmagnesiumbromide to give alcohol JJ. Alcohol JJ is then transformed intoγ,δ-unsaturated carboxylic acid DD by a three-step procedure as follows:(i) treatment with diethyl malonate and catalytic Ti(OEt)₄ at 160° C.for 1 hour, (ii) heating at 190° C. for 4 hours, and (iii) hydrolysiswith ethanolic KOH at reflux.

Carboxylic acid BB may also be prepared by General Method VIII (seeHoffman et al., Tetrahedron, 1997, 53, 7119). An amino acid KK, whichincorporates R₇ and where P₄ is an appropriate protecting group fornitrogen, is transformed into β-ketoester LL. Compound LL isdeprotonated and the resulting anion is condensed with triflate MM,which incorporates R₆. The coupling product thus obtained is treatedwith trifluoroacetic acid to provide ketoester NN, and this material issubsequently hydrolyzed to afford carboxylic acid BB. If basichydrolysis results in epimerization, ketoester NN can be transesterified(allyl alcohol, Ti(Oi-Pr)₄) and subsequently deprotected under neutralconditions (Pd(PPh₃)₄, morpholine) to give carboxylic acid BB. TriflateMM, in turn, may be prepared from the corresponding alcohol by treatmentwith trifluoromethanesulfonic anhydride and 2,6-lutidine.

Lactone AA may also be prepared by General Method IX (see: Askin et al.,J. Org. Chem. 1992, 57, 2771; and McWilliams et al., J. Am. Chem. Soc.1996, 118, 11970). An amino acid KK, which incorporates R₇ and where P₄is an appropriate protecting group for nitrogen, is transformed intoepoxide OO (single diastereomer) by the method described in Luly et al.,J. Org. Chem. 1987, 52, 1487. Epoxide OO is condensed with the anionderived from compound PP, which incorporates R₆ and in which X₂ is achiral auxiliary (including (1S,2R)-1-aminoindan-2-ol acetonide) toafford coupling product QQ. This material is subsequently cyclized underacidic conditions to provide lactone AA. Compound PP may be preparedfrom the corresponding carboxylic acid (not shown) by the methodoutlined in Askin et al., J. Org. Chem. 1992, 57, 2771.

General Method X, useful in preparation of depsipeptide compounds of theformula I-C, illustrates a method to prepare intermediate TT. Amino acidE and alcohol RR, where P₅ is an appropriate protecting group foroxygen, are subjected to an ester bond-forming reaction to produceintermediate SS. Molecule SS is deprotected to yield free carboxylicacid TT, which may be utilized in lieu of carboxylic acid K in any ofthe general methods described above.

SPECIFIC SYNTHESES

The following specific methods may also be used to prepare variouscompounds according to the invention.

Specific Method I describes the preparation of specific intermediate O1,which may be utilized as intermediate O in the general methods describedabove. Thus, ester A1 (prepared as described in Chida et al., J. Chem.Soc., Chem. Commun. 1992, 1064) is hydrolyzed to give acid B1, which, inturn, is transformed into oxazolidinone C1. Compound C1 is subsequentlydeprotonated, and the resulting enolate is diastereoselectivelyalkylated to give allyl intermediate D1. This entity is oxidized viaozonolysis, and the resulting aldehyde (not shown) is subjected to areductive amination reaction producing lactam E1. Acid-catalyzedmethanolysis of E1 then affords alcohol F1. This intermediate isoxidized via the method of Swern (or other suitable oxidationconditions) to the resulting aldehyde (not shown), which is subsequentlysubjected to an olefin-forming reaction to provide specific intermediateO1.

Specific Method II describes the preparation of specific intermediateO2, which may be utilized as intermediate O in the general methodsdescribed above. Allyl intermediate D1 is subjected to ahydroboration/oxidation sequence to afford a primary alcohol (notshown). This entity is oxidized via the method of Swern (or othersuitable oxidation conditions), and the resulting aldehyde (not shown)is subjected to a reductive-amination reaction, producing lactam G1.Acid-catalyzed methanolysis of G1 then affords alcohol H1. Thisintermediate is oxidized via the method of Swern (or other suitableoxidation conditions) to the resulting aldehyde (not shown), which issubsequently subjected to an olefin-forming reaction to provide specificintermediate O2.

The following intermediates P1, P2, and P3 may be used in the abovegeneral methods in place of intermediate O, to vary the substituentgroup in the R₂position.

A synthesis of intermediate P1 is described below. Intermediate C1(described above) is deprotonated, and the resulting enolate is trappedwith an appropriate disulfide (symmetrical or mixed) to give sulfide p1(P is a suitable protecting group for oxygen). The oxygen-protectinggroup is then removed to give alcohol p2. This intermediate is oxidizedvia the method of Swern (or using other suitable oxidation conditions),and the resulting aldehyde (not shown) is subjected to a reductiveamination reaction to give lactam p3. Acid-catalyzed methanolysis of p3then affords alcohol p4. This intermediate is oxidized via the method ofSwern (or using other suitable oxidation conditions) to the resultingaldehyde (also not shown), which is subsequently subjected to anolefin-forming reaction to provide intermediate p5. This compound may beutilized in place of intermediate O in the above general syntheticmethods; alternatively, the lactam-protecting group may be removed togive intermediate P1.

To synthesize intermediate P2, intermediate C1 is deprotonated, and theresulting enolate is trapped with an appropriate source of electrophilicoxygen (e.g., an oxaziridine) to give alcohol p6. This intermediate isalkylated with a suitably functionalized alkyl halide or triflate togive ether p7 (P is an appropriate protecting group for nitrogen). Thenitrogen-protecting group is then removed, and the resulting amine (notshown) is subjected to cyclization conditions to give lactam p8.Acid-catalyzed methanolysis of p8 then affords alcohol p9. Thisintermediate is oxidized via the method of Swern (or using othersuitable oxidation conditions) to the resulting aldehyde (also notshown), which is subsequently subjected to an olefin-forming reaction toprovide intermediate P2.

A synthesis of specific intermediate P3 is now described. IntermediateD1 (described above) is ozonized, and the resulting aldehyde (not shown)is reduced to the corresponding alcohol (also not shown). Thisintermediate is then protected to afford compound p10 (P₁ is anappropriate protecting group for oxygen). The imide functionalitypresent on p10 is hydrolyzed to carboxylic acid p11, and thisintermediate is coupled with a suitably protected hydroxylaminederivative to give amide p12 (P₂ is an appropriate protecting group foroxygen that is stable to conditions which will remove P₁). The P₁protecting group is then removed, and the resulting alcohol (p13) istransformed into an appropriate leaving group (halide or triflate, notshown). The P₂ protecting group is then removed, and the resultinghydroxamic acid is cyclized to give intermediate p14. Acid-catalyzedmethanolysis of p14 then affords alcohol p15. This intermediate isoxidized via the method of Swern (or using other suitable oxidationconditions) to the resulting aldehyde (not shown), which is subsequentlysubjected to an olefin-forming reaction to provide intermediate P3.

Specific Method III describes the preparation of intermediates Q1, Q2,and Q3, which may be utilized in the general methods described above.The known compound I1 is transformed into the literature molecule J1 bya modification of a published procedure (Ikuta et al., J. Med. Chem.1987, vol. 30, p. 1995). Independently, the amino acid ester K1 isprotected to afford silyl ether L1. The ether is reduced with DIBAL (orusing other suitable reduction conditions), and the resulting aldehyde(not shown) is subjected to an olefin-forming reaction with intermediateJ1, producing M1. Silyl deprotection of M1 then affords alcohol N1. Thisintermediate is subjected to a variety of hydrogenation conditions toprovide intermediates Q1, Q2, and Q3. These intermediates may betransformed into intermediates analogous to intermediate O1 (seeSpecific Method I above) by oxidation and subsequent olefination.

The artisan will recognize that various compounds of the invention maybe made by following the above-described general and specific methods aswell as teachings in the art, including the references cited herein, thedisclosures of which are hereby incorporated by reference. Additionally,the artisan may prepare various compounds of the invention according tothe example described below or through routine modifications to thesyntheses described herein.

EXAMPLES

Examples of various preferred compounds of formula I are set forthbelow. The structures of the compounds of the following examples wereconfirmed by one or more of the following: proton magnetic resonancespectroscopy, infrared spectroscopy, elemental microanalysis, massspectrometry, thin layer chromatography, melting-point determination,and boiling-point determination. Where there is any discrepancy betweenthe given structural formula shown for a compound and its chemical nameprovided, the structural formula applies.

Proton magnetic resonance (¹H NMR) spectra were determined using aVarian UNITYplus 300 spectrometer operating at a field strength of 300megahertz (MHz). Chemical shifts are reported in parts per million (ppm,δ) downfield from an internal tetramethylsilane standard. Alternatively,¹H NMR spectra were referenced to residual protic solvent signals asfollows: CHCl₃=7.26 ppm; DMSO=2.49 ppm; C₆HD₅=7.15 ppm. Peakmultiplicities are designated as follows: s=singlet; d=doublet;dd=doublet of doublets; t=triplet; q=quartet; br=broad resonance; andm=multiplet. Coupling constants are given in Hertz. Infrared absorption(IR) spectra were obtained using a Perkin-Elmer 1600 series FTIRspectrometer. Elemental microanalyses were performed by AtlanticMicrolab Inc. (Norcross, Ga.) and gave results for the elements statedwithin ±0.4% of the theoretical values. Flash column chromatography wasperformed using Silica gel 60 (Merck Art 9385). Analytical thin layerchromatography (TLC) was performed using precoated sheets of Silica 60F₂₅₄ (Merck Art 5719). Melting points (abbreviated as mp) weredetermined on a Mel-Temp apparatus and are uncorrected. All reactionswere performed in septum-sealed flasks under a slight positive pressureof argon, unless otherwise noted. All commercial reagents were used asreceived from their respective suppliers with the following exceptions:tetrahydrofuran (THF) was distilled from sodium-benzophenone ketyl priorto use; dichloromethane (CH₂Cl₂) was distilled from calcium hydrideprior to use; anhydrous lithium chloride was prepared by heating at 110°C. under vacuum overnight.

The following abbreviations are used herein: Et₂O refers to diethylether; DMF refers to N,N-dimethylformamide; DMSO refers todimethylsulfoxide; and MTBE refers to tert-butyl methyl ether. Otherabbreviations include: CH₃OH (methanol), EtOH (ethanol), EtOAc (ethylacetate), DME (ethylene glycol dimethyl ether), Ac (acetyl), Me(methyl), Ph (phenyl), Tr (triphenylmethyl), Cbz (benzyloxycarbonyl),Boc (tert-butoxycarbonyl), TBS (tert-butyldimethylsilyl), TFA(trifluoroacetic acid), DIEA (N,N-diisopropylethylamine), DBU(1,8-diazabicyclo[5.4.0]undec-7-ene), HOBt (1-hydroxybenzotriazolehydrate), PyBOP (benzotriazole1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate), HATU(O-(7-azabenzottiazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), EDC(1-(3-dimethylaminopropyl)-3-ethylcarbarbodiimide hydrochloride), DCC(dicyclohexylcarbodiimide), DDQ(2,3-dichloro-5,6-dicyano-1,4-benzoquinone), DMAP(4-dimethylaminopyridine), Gln (glutamine), Leu (leucine), Phe(phenylalanine), Val (valine), His (histidine), 1-Naphth(1-naphthlyalanine), 2-Naphth (2-naphthylalanine), α-t-Butyl-Gly(tert-butyl glycine), (S)-Pyrrol-Ala((2S,3′S)-2-amino-3-(2′-oxopyrrolidin-3′-yl)-propionic acid), and(S)-Piper-Ala ((2S,3′S)-2-amino-3-(2′-oxo-piperidin-3′-yl)-propionicacid). Additionally, “L” represents naturally occurring amino acids.

A simplified naming system employing amino acid abbreviations is used toidentify some intermediates and final products. When naming compounds,italicized amino acid abbreviations represent modifications at theC-terminus of that residue where the following apply: (1) acrylic acidesters are reported as “E” (trans) propenoates; (2) substituted3-methylene-dihydrofuran-2-ones are reported as “E” (trans)2-(α-vinyl-γ-butyrolactones); and (3) 5-vinylisoxazoles are reported as“E” (trans) propenisoxazoles. In addition, the terminology“AA₁Ψ[COCH₂]-AA₂” indicates that, for any peptide sequence, two aminoacids (AA₁ and AA₂) usually linked by an amide bond are replaced by aketomethlyene dipeptide isostere moiety. The terminology “AA₁-NCH₃-AA₂”indicates that, for any peptide sequence, the amide bond that usuallyconnects the two amino acids (AA₁ and AA₂) is replaced by an N-methylamide linkage. The terminology “AA₁-O-AA₂” indicates that, for anypeptide sequence, the amide bond that usually connects the two aminoacids (AA₁ and AA₂) is replaced by an ester linkage.

Examples of embodiments in accordance with the invention are describedbelow.

Example 1 Preparation of Comparison Compound #1:5-(3′-(Cbz-L-Leu-L-Phe-L-Gln)-E-Propene)-isoxazole

Preparation of Intermediate Cbz-L-(Tr-Gln)-OMe

Cbz-L-(Tr-Gln) (0.26 g, 0.50 mmol, 1 equiv.) was added to a solution ofacetyl chloride (0.25 mL, 3.52 mmol, 7.0 equiv.) in CH₃OH (5 mL), andstirring was continued at 23° C. for 1 h (hour). The solvent was removedunder reduced pressure, and the residue was dissolved in CH₂Cl₂ (100 mL)and washed with water (100 mL), saturated NaHCO₃ (100 mL), and brine(100 mL). The organic layer was dried over Na₂SO₄ and was concentrated.The residue was purified by flash column chromatography (20% EtOAc inhexanes) to afford Cbz-L-(Tr-Gln)-OMe (0.23 g, 84% yield) as a whitesolid: mp=139-140° C.; IR (cm⁻¹) 1742, 1207; ¹H NMR (DMSO-d₆) δ 1.16 (t,1H, J=7.0), 1.77 (m, 1H), 1.97 (m, 1H), 3.61 (s, 3H), 4.99 (m, 1H), 5.03(s, 2H), 7.02-7.55 (m, 20H), 7.69 (d, 1H, J=7.7), 8.59 (s, 1H); Anal.(C₃₃H₃₂N₂O₅) C, H, N.

Preparation of Intermediate Cbz-L-(Tr-Glutaminol)

Lithium chloride (0.24 g, 5.66 mmol, 2.0 equiv.) was added to a solutionof Cbz-L-(Tr-Gln)-OMe (1.50 g, 2.79 mmol, 1 equiv.) in 2:1 THF:EtOH (30mL), and the mixture was stirred at 23° C. until all solids haddissolved (10 minutes). Sodium borohydride (0.21 g, 5.55 mmol, 2.0equiv.) was added, and the reaction mixture was stirred overnight at 23°C. The solvents were removed under reduced pressure, the residue wastaken up in water (50 mL), and the pH was adjusted to 2-3 with 10% HCl.The product was extracted with EtOAc (50 mL), and the organic layer waswashed with water (50 mL) and brine (50 mL) before drying over MgSO₄.The organic layer was concentrated, and the residue was purified byflash column chromatography (gradient elution, 20→50% EtOAc in benzene)to give Cbz-L-(Tr-glutaminol) (1.02 g, 72% yield) as a white glassysolid: mp=66-70° C.; IR (cm⁻¹) 3318, 1699, 1510, 1240; ¹H NMR (DMSO-d₆)δ 1.40 (m, 1H), 1.72 (m, 1H), 2.26 (m, 2H), 3.17-3.50 (m, 3H), 4.64 (t,1H, J=5.0), 5.00 (s, 2H), 7.00-7.40 (m, 20H), 6.96 (d, 1H, J=8.5), 8.54(s, 1H); Anal. (C₃₂H₃₂N₂O₄) C, H, N.

Preparation of Intermediate L-(Tr-Glutaminol)

A suspension of Cbz-L-(Tr-glutaminol) (1.93 g, 3.79 mmol) in CH₃OH (25mL) and Pd/C (10%, 0.19 g) was stirred under a hydrogen atmosphere(balloon) for 4 hours, then was filtered through a layer of Celite. Thefiltrate was concentrated under reduced pressure to giveL-(Tr-glutaminol) as a white amorphous solid (1.38 g, 98% yield):mp=191-193° C.; IR (cm⁻¹) 3255 (br), 1642, 1527; ¹H NMR (DMSO-d₆) δ 1.29(m, 1H), 1.53 (m, 1H), 2.29 (m, 2H), 3.08 (m, 1H), 3.18 (m, 2H), 3.38(s, br, 2H), 4.43 (s, br, 1H), 7.14-7.28 (m, 15H), 8.62 (s, 1H).

Preparation of Intermediate Cbz-L-Leu-L-Phe-L-(Tr-Glutaminol)

Carbonyldiimidazole (0.17 g, 1.05 mmol, 1.0 equiv.) was added to asolution of Cbz-L-Leu-L-Phe-OH (0.41 g, 1.0 mmol, 0.95 equiv.) in THF(10 mL), and the reaction mixture was stirred at 23° C. for 1 hour.L-(Tr-Glutaminol) (0.39 g, 1.05 mmol, 1 equiv.) was then added, and theresulting solution was stirred overnight. The volatiles were removedunder reduced pressure, and the residue was purified by flash columnchromatography (gradient elution, 2→4% CH₃OH in CHCl₃) to giveCbz-L-Leu-L-Phe-L-(Tr-glutaminol) (0.47 g, 62% yield) as a whiteamorphous solid: mp=92-95° C.; IR (cm⁻¹) 3302, 1657, 1520, 1238; ¹H NMR(DMSO-d₆) δ 0.79 (t, 6H, J=7.0), 1.30 (m, 2H), 1.44 (m, 2H), 1.75 (m,1H), 2.22 (m, 2H), 2.82 (m, 1H), 2.97 (m, 1H), 3.14 (m, 1H), 3.25 (m,1H), 3.63 (m, 1H), 3.95 (m, 1H), 4.48 (m, 1H), 4.65 (t, 1H, J=5.0), 4.96(d, 1H, J=13.0), 5.02 (d, 1H, J=13.0), 7.07-7.33 (m, 25H), 7.42 (d, 1H,J=8.0), 7.66 (d, 1H, J=8.5), 7.86 (d, 1H, J=8.0), 8.52 (s, 1H); Anal.(C₄₇H₅₂N₄O₆.0.5H₂O) C, H, N.

Preparation of Intermediate Cbz-L-Leu-L-Phe-L-(Tr-Gln)-H

o-Iodoxybenzoic acid (0.63 g, 2.25 mmol, 3.0 equiv.) was added to asolution of Cbz-L-Leu-L-Phe-L-(Tr-glutaminol) (0.58 g, 0.75 mmol, 1equiv.) in DMSO (7.5 mL) at 23° C. After stirring for 2 hours, the DMSOwas removed under reduced pressure. The residue was twice diluted withCH₂Cl₂, and the solvent evaporated to remove any remaining DMSO. Theresidue was diluted with EtOAc (30 mL) and filtered, and the filtratewas washed with 5% Na₂S₂O₃/5% NaHCO₃ solution (30 mL), water (30 mL),and brine (30 mL), and then dried over Na₂SO₄. The solvent was removedunder reduced pressure to give Cbz-L-Leu-L-Phe-L-(Tr-Gln)-H (0.53 g, 92%yield) as a white glassy solid, which was used immediately withoutfurther purification: ¹H NMR (DMSO-d₆) δ 0.79 (m, 6H), 1.00-1.98 (m,5H), 2.27 (m, 2H), 2.84 (m, 1H), 3.02 (m, 1H), 3.98 (m, 2H), 4.58 (m,1H), 4.99 (s, 2H), 7.14-7.32 (m, 25H), 7.39 (d, 1H, J=8.1), 7.97 (d, 1H,J=8.5), 8.38 (d, 1H, J=8.0), 8.60 (s, 1H), 9.20 (s, 1H).

Preparation of Intermediate5-{3′-(Cbz-L-Leu-L-Phe-L-(Tr-Gln))-E-Propene}-Isoxazole

A solution of KN((CH₃)₃Si)₂ (0.95 mL of a 0.5 M solution in THF, 0.477mmol, 1.0 equiv.) was added to a suspension ofisoxazol-5-ylmethyl-triphenylphosphonium bromide (0.222 g, 0.525 mmol,1.1 equiv) in THF (20 mL) at 0° C., and the reaction was stirred at 0°C. for 30 minutes. A solution of Cbz-L-Leu-L-Phe-L-(Tr-Gln)-H (0.366 g,0.477 mmol, 1 equiv.) in THF (10 mL) was added, and the reaction mixturewas then stirred overnight at 23° C. The solvent was removed in vacuo,and the residue was diluted with EtOAc (30 mL), washed with water (30mL), and then dried over MgSO₄. The solvent was removed under reducedpressure and the residue purified by flash silica gel chromatography(gradient elution, 0→1% CH₃OH in CHCl₃) to give5-{3′-(Cbz-L-Leu-L-Phe-L-(Tr-Gln))-E-propene}-isoxazole (0.307 g, 70%yield) as an amorphous solid: IR (cm⁻¹) 3423, 1678, 1568, 1265, 1043,711; ¹H NMR (DMSO-d₆) δ 0.77-0.81 (m, 6H), 1.21-1.36 (m, 2H), 1.40-1.55(m, 1H), 1.60-1.80 (m, 2H), 2.34-2.45 (m, 2H), 2.82-2.87 (m, 1H),2.91-3.04 (m, 1H), 3.95-4.00 (m, 1H), 4.41-4.50 (m, 1H), 4.53-4.60 (m,1H), 4.99 (q, 2H, J=6.0), 6.19 (d, 1H, J=15.0), 6.36 (dd, 1H, J=15.0,6.0), 6.46 (s, 1H), 7.15-7.33 (m, 20H), 7.42 (d, 1H, J=9.0), 7.56-7.63(m, 5H), 7.96 (d, 1H, J=9.0), 8.08 (d, 1H, J=9.0), 8.51 (s, 1H), 8.58(s, 1H); HRMS calcd. for C₅₁H₅₃N₅O₆+Cs 964.3050 (M+Cs), found 964.3018.

Preparation of Product:5-(3′-(Cbz-L-Leu-L-Phe-L-Gln)-E-Propene)-Isoxazole

Trifluoroacetic acid (1 mL) was added to a solution of5-{3′-(Cbz-L-Leu-L-Phe-L-(Tr-Gln))-E-propene}-isoxazole (0.214 g, 0.257mmol) in CH₂Cl₂ (10 mL), and the reaction mixture was stirred at 23° C.overnight. The solvent was removed in vacuo and the residue purified byflash silica gel chromatography (gradient elution, 0→1% CH₃OH in CHCl₃)to give 5-(3′-(Cbz-L-Leu-L-Phe-L-Gln)-E-propene)-isoxazole as a whitesolid (0.054 g, 36% yield): ¹H NMR (DMSO-d₆) δ 0.77-0.83 (m, 6H),1.26-1.46 (m, 2H), 1.47-1.62 (m, 1H), 1.69-1.79 (m, 2H), 2.04-2.29 (m,2H), 2.83-2.88 (m, 1H), 2.97-3.10 (m, 1H), 3.99-4.12 (m, 1H), 4.37-4.43(m, 1H), 4.48-4.57 (m, 1H), 5.01 (q, 2H, J=6.0), 6.20 (d, 1H, J=15.0),6.36 (dd, 1H, J=15.0, 6.0), 6.45 (d, 1H, J=3.0), 6.75 (s, 1H), 7.14-7.29(m, 6H), 7.31-7.40 (m, 5H), 7.45 (d, 1H, J=9.0), 8.04 (d, 1H, J=9.0),8.07 (d, 1H, J=9.0), 8.51 (d, 1H, J=3.0); Anal. (C₃₂H₃₉N₅O₆) C, H, N.

Example 2 Preparation of Compound A-1:Ethyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-Propenoate

Preparation of Intermediate Boc-L-(Tr-Gln)-N(OMe)Me

Isobutyl chloroformate (4.77 mL, 36.8 mmol, 1.0 equiv.) was added to asolution of Boc-L-(Tr-Gln)-OH (18.7 g, 36.7 mmol, 1 equiv.) and4-methylmorpholine (8.08 mL, 73.5 mmol, 2.0 equiv.) in CH₂Cl₂ (250 mL)at 0° C. The reaction mixture was stirred at 0° C. for 20 min.(minutes), then N,O-dimethylhydroxylamine hydrochloride (3.60 g, 36.7mmol, 1.0 equiv.) was added. The resulting solution was stirred at 0° C.for 20 min. and at 23° C. for 2 hours (h), and then was partitionedbetween water (150 mL) and CH₂Cl₂ (2×150 mL). The combined organiclayers were dried over Na₂SO₄, and were concentrated. Purification ofthe residue by flash column chromatography (gradient elution, 40→20%hexanes in EtOAc) provided Boc-L-(Tr-Gln)-N(OMe)Me (16.1 g, 82% yield)as a white foam: IR (cm⁻¹) 3411, 3329, 3062, 1701, 1659; ¹H NMR (CDCl₃)δ 1.42 (s, 9H), 1.63-1.77 (m, 1H), 2.06-2.17 (m, 1H), 2.29-2.43 (m, 2H),3.17 (s, 3H), 3.64 (s, 4.73 (s, br, 1H), 5.38-5.41 (m, 1H), 7.20-7.31(m, 15H); Anal. (C₃₁H₃₇N₃O₅) C, H, N.

Preparation of Intermediate Boc-L-(Tr-Gln)-H

Diisobutylaluminum hydride (50.5 mL of a 1.5 M solution in toluene, 75.8mmol, 2.5 equiv.) was added to a solution of Boc-L-(Tr-Gln)-N(OMe)Me(16.1 g, 30.3 mmol, 1 equiv.) in THF at −78° C., and the reactionmixture was stirred at −78° C. for 4 hours. Methanol (4 mL) and 1.0 MHCl (10 mL) were added sequentially, and the mixture was warmed to 23°C. The resulting suspension was diluted with Et₂O (150 mL), and waswashed with 1.0 M HCl (3×100 mL), half-saturated NaHCO₃ (100 mL), andwater (100 mL). The organic layer was dried over MgSO₄, filtered, andconcentrated to give crude Boc-L-(Tr-Gln)-H (13.8 g, 97% yield) as awhite solid: mp=114-116° C.; IR (cm⁻¹) 3313, 1697, 1494; ¹H NMR (CDCl₃)δ 1.44 (s, 9H), 1.65-1.75 (m, 1H), 2.17-2.23 (m, 1H), 2.31-2.54 (m, 2H),4.11 (s, br, 1H), 5.38-5.40 (m, 1H), 7.11 (s, 1H), 7.16-7.36 (m, 15H),9.45 (s, 1H).

Preparation of Intermediate Ethyl-3-(Boc-L-(Tr-Gln))-E-Propenoate

Sodium bis(trimethylsilyl)amide (22.9 mL of a 1.0 M solution in THF,22.9 mmol, 1.0 equiv.) was added to a solution of triethylphosphonoacetate (5.59 g, 22.9 mmol, 1.0 equiv.) in THF (200 mL) at −78°C., and the resulting solution was stirred for 20 minutes at thattemperature. Crude Boc-L-(Tr-Gln)-H (10.8 g, 22.9 mmol, 1 equiv.) in THF(50 mL) was added via cannula, and the reaction mixture was stirred for2 hours at −78° C., warmed to 0° C. for 10 minutes, and then partitionedbetween 0.5 M HCl (150 mL) and a 1:1 mixture of EtOAc and hexanes (2×150mL). The combined organic layers were dried over Na₂SO₄ and wereconcentrated. Purification of the residue by flash column chromatography(40% EtOAc in hexanes) provided ethyl-3-[Boc-L-(Tr-Gln)]-E-propenoate(10.9 g, 88% yield) as a white foam: IR (cm⁻¹) 3321, 1710; ¹H NMR(CDCl₃) δ 1.27 (t, 3H, J=7.2), 1.42 (s, 9H), 1.70-1.78 (m, 1H),1.80-1.96 (m, 1H), 2.35 (t, 2H, J=7.0), 4.18 (q, 2H, J=7.2), 4.29 (s,br, 1H), 4.82-4.84 (m, 1H), 5.88 (dd, 1H, J=15.7, 1.6), 6.79 (dd, 1H,J=15.7, 5.3), 6.92 (s, 1H), 7.19-7.34 (m, 15H); Anal. (C₃₃H₃₈N₂O₅) C, H,N.

Preparation of Intermediate Ethyl-3-(Boc-L-Phe-L-(Tr-Gln))-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 15 mL), was added to a solutionof ethyl-3-(Boc-L-(Tr-Gln))-E-propenoate (3.26 g, 6.01 mmol, 1 equiv.)in the same solvent (15 mL) at 23° C. After 2 h, the volatiles wereremoved under reduced pressure to affordethyl-3-(H₂N-L-(Tr-Gln))-E-propenoate.HCl. This material was dissolvedin CH₂Cl₂ (60 mL) and Boc-L-Phe-OH (1.59 g, 6.01 mmol, 1.0 equiv.), HOBt(1.22 g, 9.02 mmol, 1.5 equiv.), 4-methylmorpholine (1.98 mL, 18.03mmol, 3 equiv.), and EDC (1.73 g, 9.02 mmol, 1.5 equiv.) were addedsequentially. The reaction mixture was stirred at 23° C. overnight, andthen was partitioned between water (100 mL) and CH₂Cl₂ (2×100 mL). Thecombined organic layers were dried over Na₂SO₄, concentrated, and theresidue was purified by flash column chromatography (40% EtOAc inhexane) to afford ethyl-3-(Boc-L-Phe-L-(Tr-Gln))-E-propenoate (3.55 g,85%) as white foam: IR (cm⁻¹) 3306, 1706, 1661; ¹H NMR (CDCl₃) δ 1.29(t, 3H, J=7.2), 1.38 (s, 9H), 1.65-1.76 (m, 1H), 1.87-1.99 (m, 1H),2.25-2.27 (m, 2H), 2.94-3.01 (m, 2H), 4.14-4.26 (m, 3H), 4.48-4.53 (m,1H), 4.95 (s, br, 1H), 5.64 (d, 1H, J=15.8), 6.29 (d, 1H, J=8.1), 6.64(dd, 1H, J=15.8, 5.4), 6.80 (s, br, 1H), 7.14-7.32 (m, 20H); Anal.(C₄₂H₄₇N₃O₆) C, H, N.

Preparation of IntermediateEthyl-3-(Boc-L-Leu-L-Phe-L-(Tr-Gln))-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) was added to a solutionof ethyl-3-(Boc-L-Phe-L-(Tr-Gln))-E-propenoate (6.40 g, 9.28 mmol, 1equiv.) in the same solvent (15 mL) at 23° C. After 2 hours, thevolatiles were removed under reduced pressure. The residue was dissolvedin CH₂Cl₂ (100 mL), and Boc-L-Leu-OH (2.58 g, 11.1 mmol, 1.2 equiv.),HOBt (1.88 g, 13.9 mmol, 1.5 equiv.), 4-methylmorpholine (3.06 mL, 27.8mmol, 3 equiv.), and EDC (2.67 g, 13.92 mmol, 1.5 equiv.) were addedsequentially. The reaction mixture was stirred at 23° C. overnight, andthen was partitioned between water (100 mL) and CH₂Cl₂ (2×100 mL). Thecombined organic layers were dried over Na₂SO₄ and concentrated, and theresidue was purified by flash column chromatography (2% CH₃OH in CH₂Cl₂)to afford ethyl-3-(Boc-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate (6.46 g, 87%yield) as white foam: IR (cm⁻¹) 3284, 1651, 1515; ¹H NMR (CDCl₃) δ 0.86(d, 3H, J=6.0), 0.89 (d, 3H, J=6.0), 1.29 (t, 3H, J=7.2), 1.34 (s, 9H),1.38-1.60 (m, 3H), 1.62-1.89 (m, 1H), 1.95-1.97 (m, 1H), 2.28-2.30 (m,2H), 3.06-3.08 (m, 2H), 3.92-3.94 (m, 1H), 4.17 (q, 2H, J=7.2),4.48-4.51 (m, 2H), 4.67 (m, 1H), 5.66 (d, 1H, J=15.9), 6.51-6.57 (m,2H), 6.69 (dd, 1H, J=15.6, 5.1), 7.10-7.33 (m, 21H); Anal.(C₄₈H₅₈N₄O₇.0.33H₂O) C, H, N.

Preparation of IntermediateEthyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 3 mL), was added to a solutionof ethyl-3-(Boc-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate (0.216 g, 0.27mmol, 1 equiv.) in the same solvent (3 mL) at 23° C. After 2 hours, thevolatiles were removed under reduced pressure. The residue was dissolvedin CH₂Cl₂ (15 mL), cooled to 0° C., and triethylamine (0.112 mL, 0.81mmol, 3.0 equiv.) and 5-methylisoxazole-3-carbonyl chloride (0.058 g,0.40 mmol, 1.5 equiv.) were added sequentially. The reaction mixture wasstirred at 0° C. for 30 minutes, and then was partitioned between water(50 mL) and CH₂Cl₂ (2×50 mL). The combined organic layers were driedover Na₂SO₄ and concentrated, and the residue was purified by flashcolumn chromatography (2% CH₃OH in CH₂Cl₂) to affordethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate(0.199 g, 91% yield) as a white foam: IR (cm⁻¹) 3286, 1650, 1541; ¹H NMR(CDCl₃) δ 0.86 (d, 3H, J=5.4), 0.89 (d, 3H, J=5.7 ), 1.28 (t, 3H,J=7.2), 1.43-1.59 (m, 2H), 1.67-1.75 (m, 1H), 1.95-1.99 (m, 2H), 2.28(t, 2H, J=7.2), 2.41 (s, 3H), 2.97-3.04 (m, 1H), 3.06-3.13 (m, 1H), 4.17(q, 2H, J=7.2), 4.31-4.33 (m, 1H), 4.48-4.52 (m, 2H), 5.72 (d, 1H,J=15.9), 6.19 (s, 1H), 6.41 (d, 1H, J=7.5), 6.59 (d, 1H, J=8.1), 6.71(dd, 1H, J=15.3, 6.0), 6.95 (d, 1H, J=6.6), 7.09-7.21 (m, 21H); Anal.(C₄₈H₅₃N₅O₇.H₂O) C, H, N.

Preparation of ProductEthyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-Propenoate

Triisopropylsilane (0.077 mL, 0.376 mmol, 1.8 equiv.) andtrifluoroacetic acid (3 mL) were added sequentially to a solution ofethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate(0.185 g, 0.21 mmol, 1 equiv.) in CH₂Cl₂ (3 mL) at 23° C., producing abright yellow solution. The reaction mixture was stirred for 30 minutesat 23° C., during which time it became colorless. The volatiles wereremoved under reduced pressure, and the resulting white solid wastriturated with Et₂O (10 mL), filtered, and air-dried to giveethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate(0.87 g, 81% yield) as white solid: mp=223-225° C.; IR (cm⁻¹) 3298,1662, 1544, 1457, 1278; ¹H NMR (DMSO-d₆) δ 0.81 (d, 3H, J=6.0 ), 0.85(d, 3H, J=6.3), 1.23 (t, 3H, J=6.9), 1.38-1.42 (m, 1H), 1.48-1.77 (m,4H), 2.04 (t, 2H, J=7.2), 2.46 (s, 3H), 2.78-2.86 (m, 1H), 2.93-3.00 (m,1H), 4.11 (q, 2H, J=7.2), 4.36-4.54 (m, 3H), 5.63 (d, 1H, J=15.6), 6.56(s, 1H), 6.68 (dd, 1H, J=15.9, 5.4), 6.76 (s, br, 1H), 7.19 (m, 6H),8.09 (d, 1H, J=8.1), 8.14 (d, 1H, J=7.8), 8.58 (d, 1H, J=7.5); Anal.(C₂₉H₃₉N₅O₇) C, H, N.

Example 3 Preparation of Compound A-2:Ethyl-3-((Isoxazole-5′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-Propenoate

Preparation of IntermediateEthyl-3-((Isoxazole-5′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-Propenoate

This compound was prepared fromethyl-3-(Boc-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate andisoxazole-5-carbonyl chloride using the procedure described above(Example 2) for the preparation ofethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate:IR (cm⁻¹) 3282, 1643, 1530; ¹H NMR (CDCl₃) δ 0.87 (t, 6H, J=6.6), 1.29(t, 3H, J=7.2), 1.49-1.64 (m, 3H), 1.69-1.80 (m, 1H), 1.90-1.96 (m, 1H),2.30 (t, 2H, J=7.2), 2.92-2.96 (m, 1H), 3.02-3.09 (m, 1H), 4.17 (q, 2H,J=7.2), 4.42-4.48 (m, 3H), 5.69 (d, 1H, J=15.3), 6.65 (s, br, 1H), 6.66(dd, 1H, J=15.9, 5.4), 6.76-6.79 (m, 2H), 7.00-7.31 (m, 22H), 8.24 (s,1H); Anal. (C₄₇H₅₁N₅O₇.0.75H₂O) C, H, N.

Preparation ofEthyl-3-((Isoxazole-5′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-Propenoate

The title compound was prepared fromethyl-3-((isoxazole-5′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoateusing a procedure analogous to that described above (Example 2) for thepreparation ofethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate:mp=217-220° C.; IR (cm⁻¹) 3302, 1655, 1541; ¹H NMR (DMSO-d₆) δ 0.81 (d,3H, J=6.0), 0.86 (d, 3H, J=6.0), 1.21 (t, 3H, J=7.2), 1.42-1.75 (m, 5H),2.04 (t, 2H, J=7.2), 2.78-2.87 (m, 1H), 2.94-3.01 (m, 1H), 4.11 (q, 2H,J=7.2), 4.37 (m, 1H), 4.41-4.52 (m, 2H), 5.64 (d, 1H, J=15.6), 6.68 (dd,1H, J=15.9, 5.4), 6.76 (s, br, 1H), 7.12-7.19 (m, 7H), 8.02 (d, 1H,J=8.1), 8.20 (d, 1H, J=8.1), 8.74 (d, 1H, J=1.8), 8.94 (d, 1H, J=7.8);Anal. (C₂₈H₃₇N₅O₇) C, H, N.

Example 4 Preparation of Compound A-3:Ethyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-Gln)-E-Propenoate

Preparation of IntermediateEthyl-3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-Propenoate

Ethyl-3-(H₂N-L-(Tr-Gln))-E-propenoate.HCl (prepared as described inExample 2 above, 1.37 g, 3.10 mmol) was dissolved in DMF (10 mL) at 23°C. Diisopropylethylamine (1.08 mL, 6.20 mmol) was added, followed byBoc-L-(4-Me-Phe)-OH (0.87 g 3.10 mmol). The reaction was cooled to 0° C.HATU (1.18 g, 3.10 mmol) was added, and the reaction allowed to warm toroom temperature. The DMF was removed in vacuo. The residue wasdissolved in EtOAc (30 mL), and the organic phase was washedsequentially with 10% HCl solution (25 mL), saturated NaHCO₃ solution(25 mL), H₂O (25 mL), and brine (25 mL). The solvent was dried (MgSO₄)and filtered, and the residue purified by flash column chromatography(gradient elution, 0→0.75% CH₃OH in CHCl₃) to giveethyl-3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate (1.48 g, 68% yield)as a white amorphous solid: IR (cm⁻¹) 1713, 1655, 1491, 1175; ¹H NMR(DMSO-d₆) δ 1.20 (t, 3H, J=7.0), 1.30 (s, 9H), 1.62-1.66 (m, 2H), 2.23(s, 3H), 2.32 (m, 2H), 2.72 (m, 1H), 2.84 (m, 1H), 4.07-4.09 (m, 1H),4.10 (q, 2H, J=7.0), 4.38 (m, 1H), 5.64 (d, 1H, J=15.5), 6.72 (dd, 1H,J=15.5, 5.5), 6.88 (d, 1H, J=8.0), 7.04 (d, 2H, J=7.7), 7.10 (d, 2H,J=7.7), 7.14-7.28 (m, 15H), 8.02 (d, 1H, J=8.0), 8.53 (s, 1H); Anal.(C₄₃H₄₉N₃O₆) C, H, N.

Preparation of IntermediateEthyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-Propenoate

Ethyl-3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate (1.45 g, 2.06 mmol)was dissolved in 1,4-dioxane (27 mL), and a solution of HCl in1,4-dioxane (4.0 M, 14 mL) was added. The reaction was stirred at roomtemperature for 4 hours. The solvent was removed by evaporation, and theresidue taken up in EtOAc (50 mL). The organic phase was washed withsaturated NaHCO₃ solution (50 mL) and then brine (50 mL), dried (MgSO₄),and the solvent removed under reduced pressure to give 1.23 g of anoff-white amorphous solid. This material was coupled withBoc-L-α-(t-Butyl-Gly)-OH using the procedure described for the synthesisof ethyl-3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate above to affordethyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate(49% yield) as a white amorphous solid: IR (cm⁻¹) 1655, 1507, 1248,1171; ¹H NMR (DMSO-d₆) δ 0.81 (s, 9H), 1.21 (t, 3H, J=7.0), 1.37 (s,9H), 1.52-1.70 (m, 2H), 2.22 (s, 3H), 2.26-2.28 (m, 2H), 2.73-2.91 (m,2H), 3.86 (d, 1H, J=9.6), 4.05-4.14 (m, 2H), 4.31-4.36 (m, 1H),4.47-4.55 (m, 1H), 5.54 (d, 1H, J=15.4), 6.37 (d, 1H, J=9.6), 6.65 (dd,1H, J=15.8, 5.5), 7.01 (d, 2H, J=8.1), 7.07 (d, 2H, J=7.7), 7.11-7.32(m, 15H), 8.03 (d, 1H, J=8.1), 8.10 (d, 1H, J=7.7), 8.49 (s, 1H); Anal.(C₄₉H₆₀N₄O₇.0.4H₂O) C, H, N.

Preparation of IntermediateEthyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-Propenoate

Ethyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate wasdeprotected using the procedure described for the deprotection ofethyl-3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate above, and theresulting amine (0.22 g, 0.30 mmol) was dissolved in CH₂Cl₂ (3 mL).Pyridine (0.025 mL, 0.32 mmol) was added, and the reaction was cooled to0° C. 5-Methylisoxazole-3-carbonyl chloride (0.046 g, 0.32 mmol) wasadded. The reaction was allowed to warm to room temperature, and wasstirred for one hour. The solvent was removed in vacuo, and the residuesubjected to flash column chromatography (gradient elution, 0→1% CH₃OHin CH₂Cl₂) to affordethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate(0.19 g, 77% yield) as a white amorphous solid: IR (cm⁻¹) 1651, 1518; ¹HNMR (DMSO-d₆) δ 0.88 (s, 9H), 1.20 (t, 3H, J=7.0), 1.55-1.67 (m, 2H),2.14 (s, 3H), 2.18-2.28 (m, 2H), 2.45 (s, 3H), 2.70-2.77 (m, 1H),2.86-2.93 (m, 1H), 4.07-4.14 (m, 2H), 4.46 (d, 1H, J=9.6), 4.50-4.55 (m,1H), 5.54 (d, 1H, J=15.8), 6.59 (s, 1H), 6.65 (dd, 1H, J=15.8, 5.5,15.8), 6.95 (d, 2H, J=8.1), 7.05 (d, 2H, J=8.1), 7.13-7.28 (m, 15H),7.60 (d, 1H, J=9.6), 8.13 (d, 1H, J=8.1), 8.41 (d, 1H, J=8.1), 8.51 (s,1H); Anal. (C₄₉H₅₅N₅O₇) C, H, N.

Preparation of ProductEthyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-Gln)-E-Propenoate

Ethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate(0.17 g, 0.20 mmol) was dissolved in CH₂Cl₂ (4 mL) at 23° C.Trifluoroacetic acid (0.4 mL) was added, and the reaction was stirred atroom temperature for six hours. The solvents were removed in vacuo, andthe residue was subjected to flash column chromatography (gradientelution, 0→2% CH₃OH in CH₂Cl₂) to affordethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-Gln)-E-propenoate(0.085 g, 73% yield) as a white amorphous solid: IR (cm⁻¹) 1661, 1541,1206; ¹H NMR (DMSO-d₆) δ 0.88 (s, 9H), 1.21 (t, 3H, J=7.0), 1.60-1.73(m, 2H, 2.01-2.06 (m, 2H), 2.14 (s, 3H), 2.50 (s, 3H), 2.70-2.77 (m,1H), 2.86-2.93 (m, 1H), 4.07-4.14 (m, 2H), 4.34-4.37 (m, 1H), 4.45 (d,1H, J=9.6), 4.50-4.55 (m, 1H), 5.57 (d, 1H, J=15.8), 6.60 (s, 1H), 6.66(dd, 1H, J=15.8, 5.5), 6.75 (s, br, 1H), 6.96 (d, 2H, J=8.1), 7.06 (d,2H, J=7.7), 7.17 (s, br, 1H), 7.65 (d, 1H, J=9.6), 8.14 (d, 1H, J=8.1),8.40 (d, 1H, J=7.7); Anal. (C₃₀H₄₁N₅O₇.0.5TFA.0.5H₂O) C, H, N.

Example 5 Preparation of Compound A-4:Ethyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-Gln)-E-Propenoate

Preparation of IntermediateEthyl-3-(Boc-L-(4-F-Phe)-L-(Tr-Gln))-E-Propenoate

Boc-L-(4-F-Phe)-OH (1.41 g, 5.0 mmol) was dissolved in THF (50 mL).Ethyl-3-(H₂N-L-(Tr-Gln))-E-propenoate.HCl (prepared as described inExample 2 above, 1.0 g, 5.0 mmol) was added, followed by Et₃N (0.70 mL,5.0 mmol). Carbonyldiimidazole (0.81 g, 5.0 mmol) was added, and thereaction was stirred at room temperature for 20 hours. The solvent wasremoved in vacuo, and the residue subjected to flash columnchromatography (gradient elution, 0→1% CH₃OH in CH₂Cl₂) to affordethyl-3-(Boc-L-(4-F-Phe)-L-(Tr-Gln))-E-propenoate (1.13 g, 32% yield) asa white amorphous solid: IR (cm⁻¹) 1712, 1666, 1510, 1169; ¹H NMR(DMSO-d₆) δ 1.20 (t, 3H, J=7.0), 1.29 (s, 9H), 1.61-1.70 (m, 2H),2.27-2.34 (m, 2H), 2.74-2.78 (m, 1H), 2.86-2.90 (m, 1H), 4.06-4.13 (m,3H), 4.36-4.40 (m, 1H), 5.58 (d, 1H, J=15.6), 6.71 (dd, 1H, J=15.6,5.5), 6.98 (d, 1H, J=8.1), 7.03-7.09 (m, 2H), 7.14-7.28 (m, 17H), 8.06(d, 1H, J=8.1), 8.53 (s, 1H); LRMS (M+Na) 730.

Preparation of IntermediateEthyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-E-Propenoate

Ethyl-3-(Boc-L-(4-F-Phe)-L-(Tr-Gln))-E-propenoate was deprotected andcoupled with Boc-L-α-(t-Butyl-Gly)-OH using the procedures describedabove to prepareethyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate, toprovideethyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-E-propenoate (54%yield) as a white amorphous solid: IR (cm⁻¹) 1720, 1651, 1506, 1168; ¹HNMR (DMSO-d₆) δ 0.80 (s, 9H), 1.20 (t, 3H, J=7.0), 1.36 (s, 9H),1.53-1.67 (m, 2H), 2.23-2.28 (m, 2H), 2.79-2.94 (m, 2H), 3.85 (d, 1H,J=9.9), 4.09 (q, 2H, J=7.0), 4.31-4.35 (m, 1H), 4.53-4.55 (m, 1H), 5.46(d, 1H, J=15.8), 6.36 (d, 1H, J=9.2), 6.64 (dd, 1H, J=15.8, 5.5),6.97-7.03 (m, 2H), 7.13-7.28 (m, 17H), 8.08 (d, 1H, J=8.1), 8.14 (d, 1H,J=8.1), 8.49 (s, 1H); Anal. (C₄₈H₅₇N₄O₇F) C, H, N.

Preparation of IntermediateEthyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-E-Propenoate

Ethyl-3-(Boc-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-E-propenoate wasdeprotected and coupled with 5-methylisoxazole-3-carbonyl chloride usingthe procedures described above to prepareethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoateto affordethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-E-propenoate(74% yield) as a white amorphous solid: IR (cm⁻¹) 1659, 1535, 1510; ¹HNMR (DMSO-d₆) δ 0.88 (s, 9H), 1.20 (t, 3H, J=7.4), 1.52-1.67 (m, 2H),2.23-2.28 (m, 2H), 2.45 (s, 3H), 2.75-2.82 (m, 1H), 2.89-2.96 (m, 1H),4.09 (q, 2H, J=7.0), 4.32-4.36 (m, 1H), 4.45 (d, 1H, J=9.6), 4.50-4.55(m, 1H), 5.44 (d, 1H, J=15.6), 6.58 (s, 1H), 6.63 (dd, 1H, J=15.6, 5.5,15.6), 6.93-6.99 (m, 2H), 7.13-7.28 (m, 17H), 7.64 (d, 1H, J=9.6), 8.16(d, 1H, J=8.5), 8.46 (d, 1H, J=8.1), 8.51 (s, 1H); Anal. (C₄₈H₅₂N₅O₇F)C, H, N.

Preparation ofEthyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-Gln)-E-Propenoate

Ethyl-3-[(5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln)]-E-propenoatewas deprotected using a procedure analogous to that described above forthe preparation ofethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-Me-Phe)-L-Gln)-E-propenoateto provideethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-Gln)-E-propenoate(80% yield) as a white amorphous solid: IR (cm⁻¹) 1653, 1543, 1223; ¹HNMR (DMSO-d₆) δ 0.88 (s, 9H), 1.21 (t, 3H, J=7.0), 1.59-1.75 (m, 2H),2.01-2.06 (m, 2H), 2.46 (s, 3H), 2.75-2.82 (m, 1H), 2.89-2.96 (m, 1H),4.09 (q, 2H, J=7.0), 4.33-4.36 (m, 1H), 4.44 (d, 1H, J=9.6), 4.50-4.58(m, 1H), 5.47 (d, 1H, J=15.8), 6.59 (s, 1H), 6.64 (dd, 1H, J=15.8, 5.5),6.75 (s, br, 1H), 6.94-7.00 (m, 2H), 7.16 (s, br, 1H), 7.18-7.23 (m,2H), 7.69 (d, 1H, J=9.6), 8.16 (d, 1H, J=8.1), 8.44 (d, 1H, J=8.1);Anal. (C₂₉H₃₈N₅O₇F) C, H, N.

Example 6 Preparation of Compound A-5:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

Preparation of Intermediate(4S)-4-(2′-Carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic Acidtert-Butyl Ester

Sodium hydroxide (27 mL of a 4.0 M solution in H₂O, 108 mmol, 3.0equiv.) was added to a solution of(4S)-4-(2-methoxycarbonylethyl)-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-but ester (prepared as described in Chida et al., J. Chem.Soc., Chem. Commun. 1992, 1064) (10.5 g, 36.5 mmol, 1 equiv.) in CH₃OH(150 mL), and the resulting cloudy reaction mixture was stirred at 23°C. for 3.5 h. The mixture was concentrated under reduced pressure to ˜30mL volume, and then was partitioned between 0.5 M HCl (150 mL) and EtOAc(2×150 mL). The combined organic layers were dried over MgSO₄ and weregravity filtered. The filtrate was concentrated under reduced pressureand the residue dried under vacuum, to afford(4S)-4-(2′-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acidtert-butyl ester (10.0 g, 100% crude yield). This material was usedwithout further purification: 1H NMR (CDCl₃, mixture of rotamers) δ 1.49(s), 1.57 (s), 1.60 (s), 1.84-2.05 (m), 2.39-2.41 (m), 3.71-3.74 (m),3.91-4.05 (m).

Preparation of Intermediate(4S,4″S)-4-{3′-(4″-Benzyl-2″-oxo-oxazolidin-3″-yl)-3′-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylicAcid tert-Butyl Ester

Triethylamine (8.87 mL, 63.6 mmol, 3.0 equiv.) and pivaloyl chloride(2.61 mL, 21.2 mmol, 1.0 equiv.) were added sequentially to a solutionof (4S)-4-(2′-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acidtert-butyl ester (5.80 g, 21.2 mmol, 1 equiv.) in THF (450 mL) at 0° C.The cloudy reaction mixture was stirred at 0° C. for 3.5 h, then lithiumchloride (0.988 g, 23.3 mmol, 1.1 equiv.) and(S)-(−)-4-benzyl-2-oxazolidinone (3.57 g, 20.1 mmol, 0.95 equiv.) wereadded sequentially. After warming to 23° C. and stirring for 19 h, thereaction mixture was partitioned between 0.5 M HCl (150 mL) and EtOAc(2×150 mL). The combined organic layers were washed with half-saturatedNa₂CO₃ (150 mL), dried over MgSO₄, and gravity filtered. The filtratewas concentrated under reduced pressure and the residue was purified byflash column chromatography (30% EtOAc in hexanes) to give(4S,4″S)-4-{3′-(4″-benzyl-2″-oxo-oxazolidin-3″-yl)-3′-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (7.17 g, 83%) as a colorless oil: IR (cm⁻¹) 2978,1783, 1694; ¹H NMR (CDCl₃, mixture of rotamers) δ 1.49 (s), 1.59 (s),1.63 (s), 2.01-2.10 (m), 2.76 (dd, J=13.5, 9.8), 2.82-3.13 (m),3.30-3.41 (m), 3.76-3.82 (m), 3.90 (s, br), 3.97 (dd, J=9.0, 5.6),4.10-4.19 (m), 4.63-4.71 (m), 7.22-7.36 (m); Anal. (C₂₃H₃₂N₂O₆) C, H, N.

Preparation of Intermediate(2′S,4S,4″S)-4-{2′-(4″-Benzyl-2″-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl}-2,2-dimethyloxazolidine-3-carboxylicAcid tert-Butyl Ester

A solution of(4S,4″S)-4-{3′-(4″-benzyl-2″-oxo-oxazolidin-3″-yl)-3′-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (7.17 g, 16.6 mmol, 1 equiv.) in THF (50 mL) wasadded to a solution of sodium bis(trimethylsilyl)amide (16.6 mL of a 1.0M solution in THF, 16.6 mmol, 1.0 equiv.) in the same solvent (150 mL)at −78° C. The reaction mixture was stirred for 20 min. at −78° C., andthen allyl iodide (4.55 mL, 49.8 mmol, 3.0 equiv.) was added. Afterstirring an additional 3 h at −78° C., the reaction mixture wasmaintained at −45° C. for 2 h, and then was partitioned between a 2:1mixture of half-saturated NH₄Cl and 5% Na₂S₂O₃ (300 mL) and a 1:1mixture of EtOAc and hexanes (2×200 mL). The combined organic layerswere washed with H₂O (200 mL), dried over MgSO₄, and gravity filtered.The filtrate was concentrated under reduced pressure and the residue waspurified by flash column chromatography (15% EtOAc in hexanes) toprovide(2′S,4S,4″S)-4-{2′-(4″-benzyl-2″-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (4.29 g, 55%) as a colorless oil: IR (cm⁻¹) 2978,1780, 1695; ¹H NMR (CDCl₃, mixture of rotamers) δ 1.45 (s), 1.49 (s),1.68-1.80 (m), 2.13-2.47 (m), 2.49-2.67 (m), 3.32 (dd, J=13.4, 3.1),3.69-3.97 (m), 4.11-4.21 (m), 4.66-4.74 (m), 5.06-5.13 (m), 5.74-5.88(m), 7.20-7.36 (m); Anal. (C₂₆H₃₆N₂O₆) C, H, N.

Preparation of Intermediate(1S,3S)-{3-(1′-(2″,4″-Dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylpropyl}-carbamicAcid tert-Butyl Ester

Ozone was bubbled through a solution of(2′S,4S,4″S)-4-(2′-(4″-benzyl-2″-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl)-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (4.29 g, 9.08 mmol, 1 equiv.) in CH₂Cl₂ (200 mL)and CH₃OH (0.735 mL, 18.1 mmol, 2.0 equiv.) at −78° C. until a bluecolor persisted. The reaction mixture was then purged with argon untilit became colorless. Methyl sulfide (6.67 mL, 90.8 mmol, 10 equiv.) wasadded, the mixture was stirred at −78° C. for 3.5 h, and was maintainedat 0° C. for an additional 1 h. After partitioning the reaction mixturebetween H₂O (200 mL) and a 1:1 mixture of EtOAc and hexanes (2×200 mL),the combined organic layers were dried over MgSO₄ and gravity filtered.The filtrate was concentrated under reduced pressure and the residue wasimmediately utilized without further purification.

The above residue was dissolved in a 2:1 mixture of THF and EtOH (240mL) at 23° C., and 2,4-dimethoxybenzylamine hydrochloride (7.40 g, 36.3mmol, 4.0 equiv.), sodium acetate (2.98 g, 36.2 mmol, 4.0 equiv.), andsodium cyanoborohydride (1.14 g, 18.1 mmol, 2.0 equiv.) were addedsequentially. The resulting suspension was stirred for 18 h at 23° C.,and then was partitioned between 0.5 M HCl (400 mL) and EtOAc (2×200mL). The combined organic layers were washed with half-saturated NaHCO₃(300 mL), dried over Na₂SO₄, and concentrated under reduced pressure.The residue was passed through a short silica gel column (eluting with50% EtOAc in hexanes) to give(3′S,4S)-4-{1′-(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-ylmethyl}-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester contaminated with(S)-(−)-4-benzyl-2-oxazolidinone.

This material was dissolved in CH₃OH (100 mL), and TsOH.H₂O (0.345 g,1.81 mmol, 0.20 equiv.) was added. The reaction mixture was heated to50° C., and was maintained at that temperature for 2.5 h. After coolingto 23° C., the reaction mixture was concentrated under reduced pressureto ˜20 mL volume and was partitioned between half-saturated NaHCO₃ (150mL) and a 9:1 mixture of CH₂Cl₂ and CH₃OH (2×150 mL). The combinedorganic layers were dried over Na₂SO₄ and concentrated under reducedpressure. Purification of the residue by flash column chromatography (3%CH₃OH in CH₂Cl₂) afforded(1S,3′S)-{2-(1′-(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylethyl}-carbamicacid tert-butyl ester (1.62 g, 44%) as a foam: IR (cm⁻¹) 3328, 1669; ¹HNMR (CDCl₃) δ 1.44 (s, 9H), 1.50-1.75 (m, 2H), 1.90-2.00 (m, 1H),2.17-2.27 (m, 1H), 2.52-2.62 (m, 1H), 3.14-3.24 (m, 2H), 3.51-3.65 (m,3H), 3.70-3.78 (m, 1H), 3.80 (s, 6H), 4.35 (d, 1H, J=14.3), 4.48 (d, 1H,J=14.3), 5.51-5.54 (m, 1H), 6.42-6.46 (m, 2H), 7.09-7.12 (m, 1H); Anal.(C₂₁H₃₂N₂O₆) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala])-E-Propenoate

DMSO (0.270 mL, 3.80 mmol, 3 equiv. was added dropwise to a −78° C.solution of oxalyl chloride (0.166 mL, 1.90 mmol, 1.5 equiv.) in CH₂Cl₂(14 mL). The reaction mixture was stirred 20 min., then a solution of(1S,3′S)-{2-(1′-(2″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylethyl}-carbamicacid tert-butyl ester (0.518 g, 1.27 mmol, 1 equiv.) in CH₂Cl₂ (13 mL)was added via cannula along the side of the reaction vessel. Afterstirring 20 min., triethylamine (1.06 mL, 7.60 mmol, 6 equiv.) was addeddropwise, and the reaction mixture was stirred for 1.5 h. Acetic acid(0.479 mL, 8.37 mmol, 6.6 equiv.) was added, and the reaction mixturewas warmed to 0° C. for 5 min., then diluted with MTBE (200 mL) andwashed with water, saturated NaHCO₃, and brine (25 mL each). The organicphase was dried over Na₂SO₄ and concentrated to provide the crudealdehyde as a foam (0.516 g, quant.), which was used without furtherpurification.

Sodium bis(trimethylsilyl)amide (1.23 mL of a 1.0 M solution in TEF,1.23 mmol, 1 equiv.) was added to a solution of triethylphosphonoacetate (0.244 mL, 1.23 mmol, 1 equiv.) in THF (15 mL) at −78°C., and the resulting solution was stirred for 20 min. at thattemperature. The crude aldehyde (prepared above, 0.500 g, 1.23 mmol, 1equiv.) in THF (13 mL) was added via cannula along the side of thereaction vessel, and the reaction mixture was stirred for 45 min. at−78° C., warmed to 0° C. for 7 min., and partitioned between 0.5 M HCl(20 mL) and MTBE (2×50 mL). The combined organic layers were dried overMgSO₄ and were concentrated. Purification of the residue by flash columnchromatography (60% EtOAc in hexanes) providedethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate(0.356 g, 61%) as a white foam: R_(f)=0.43 (60% EtOAc in hexanes); IR(cm⁻¹) 3307, 1708, 1678; ¹H NMR (CDCl₃) δ 1.28 (t, 3H, J=7.2), 1.43 (s,9H), 1.52-1.70 (m, 2H), 1.98-2.09 (m, 1H), 2.21-2.34 (m, 1H), 2.48-2.59(m, 1H), 3.16-3.24 (m, 2H), 3.80 (s, 6H), 4.18 (q, 2H, J=7.2), 4.27-4.40(m, 1H), 4.41 (s, 2H), 5.40 (d, 1H, J=8.1), 5.95 (dd, 1H, J=15.6, 1.6),6.41-6.48 (m, 2H), 6.86 (dd, 1H, J=15.6, 5.3), 7.08-7.13 (m, 1H); Anal.(C₂₅H₃₆N₂O₇.0.25H₂O) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-(4-F-Phe)-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-Propenoate

This material was prepared fromethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoateand Boc-L-(4-F-Phe)-OH using a procedure similar to that described forthe preparation of ethyl-3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate(Example 4) above: R_(f)=0.34 (60% EtOAc in hexanes); IR (cm⁻¹) 3258,1705, 1666; ¹H NMR (CDCl₃) δ 1.28 (t, 3H, J=7.2), 1.45 (s, 9H),1.51-1.66 (m, 2H), 1.78-1.90 (m, 1H), 2.06-2.23 (m, 2H), 2.99 (dd, 1H,J=13.7, 6.2), 3.11 (dd, 1H, J=13.7, 5.3), 3.17-3.23 (m, 2H), 3.80 (s,3H), 3.81 (s, 3H), 4.18 (q, 2H, J=7.2), 4.35 (s, 2H), 4.38-4.51 (m, 2H),5.29-5.37 (m, 1H), 5.76 (d, 1H, J=15.8), 6.43-6.47 (m, 2H), 6.72 (dd,1H, J=15.8, 5.3), 6.83-6.91 (m, 2H), 7.09-7.17 (m, 3H), 7.92 (br, 1H);Anal. (C₃₄H₄₄FN₃O₈) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-Val-L-(4-F-Phe)-L-[(N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala]}-E-Propenoate

This compound was prepared fromethyl-3-{Boc-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoateand Boc-L-Val-OH using a similar procedure to that described above forthe preparation of ethyl-3-(Boc-L-Phe-L-(Tr-Gln))-E-propenoate (Example2): R_(f)=0.24 (60% EtOAc in hexanes); IR (cm⁻¹) 3284, 1713, 1678 br,1643; ¹H NMR (CDCl₃) δ 0.91 (d, 3H, J=6.8), 0.97 (d, 3H, J=6.8), 1.28(t, 3H, J=7.2), 1.45 (s, 9H), 1.50-1.62 (m, 2H), 1.66-1.82 (m, 1H),1.90-2.02 (m, 1H), 2.08-2.21 (m, 2H), 2.94 (dd, 1H, J=13.5, 5.8),3.17-3.27 (m, 3H), 3.80(s, 3H), 3.82 (s, 3H), 3.97-4.05 (m, 1H), 4.17(q, 2H, J=7.2), 4.27 (d, 1H, J=14.3), 4.29-4.38 (m, 1H), 4.40 (d, 1H,J=14.3), 4.86-4.93 (m, 1H), 5.10 (d, 1H, J=8.7), 5.76 (dd, 1H, J=15.6,1.2), 6.45-6.52 (m, 2H), 6.70 (dd, 1H, J=15.6, 5.4), 6.79-6.88 (m, 3H),7.12-7.22 (m, 3H), 8.30 (d, 1H, J=5.9); Anal. (C₃₉H₅₃FN₄O₉) C, H, N.

Preparation of IntermediateEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-Propenoate

This compound was prepared fromethyl-3-{Boc-L-Val-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoateand isoxazole-5-carbonyl chloride using the procedure described above(Example 2) for the preparation ofethyl-3-((5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate:R_(f)=0.36 (5% CH₃OH in CH₂Cl₂); IR (cm⁻¹) 3284, 1717, 1650; ¹H NMR(CDCl₃) δ 0.97 (d, 3H, J=6.8), 1.01 (d, 3H, J=6.8), 1.28 (t, 3H, J=7.2),1.51-1.64 (m, 2H), 1.72-1.84 (m, 1H), 1.95-2.05 (m, 1H), 2.11-2.33 (m,2H), 2.48 (s, 3H), 2.98 (dd, 1H, J=13.7, 5.6), 3.16-3.24 (m, 3H), 3.80(s, 3H), 3.81 (s, 3H), 4.17 (q, 2H, J=7.2), 4.23 (d, 1H, J=14.3),4.31-4.42 (m, 1H), 4.40 (d, 1H, J=14.3), 4.44-4.50 (m, 1H), 4.88-4.96(m, 1H), 5.79 (dd, 1H, J=15.6, 1.4), 6.43-6.49 (m, 3H), 6.71 (dd, 1H,J=15.6, 5.3), 6.80-6.88 (m, 2H), 6.94 (d, 1H, J=9.3), 7.11-7.17 (m, 3H),7.29 (d, 1H, J=8.7), 8.33 (d, 1H, J=6.2); Anal. (C₃₉H₄₈FN₅O₉.0.5H₂O) C,H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

A suspension ofethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate(0.263 g, 0.351 mmol, 1 equiv.), water (2 drops), and DDQ (0.104 g,0.458 mmol, 1.3 equiv.) was refluxed for 9 h and then allowed to cool toroom temperature over 8 h. The reaction mixture was diluted with CH₂Cl₂(200 mL) and washed with a 2:1 mixture of saturated NaHCO₃ and 1 N NaOH(20 mL). The organic phase was dried over MgSO₄ and evaporated.Purification of the residue by flash column chromatography (gradientelution 2→3% CH₃OH in CH₂Cl₂) gaveethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-pyrrol-Ala)}-E-propenoate(0.117 g, 56%) as a white solid: mp=219-220° C.; R_(f)=0.23 (5% CH₃OH inCH₂Cl₂); IR (cm⁻¹) 3401 br, 3295, 1655 br; ¹H NMR (CDCl₃) δ 0.94 (d, 3H,J=6.8), 0.97 (d, 3H, J=6.5), 1.29 (t, 3H, J=7.2), 1.54-1.65 (m, 1H),1.72-1.91 (m, 2H), 2.07-2.26 (m, 2H), 2.28-2.39 (m, 1H), 2.49 (d, 3H,J=0.9), 3.01 (dd, 1H, J=13.8, 6.1), 3.12 (dd, 1H, J=13.8, 6.4),3.26-3.38 (m, 2H), 4.18 (q, 2H, J=7.2), 4.34 (dd, 1H, J=8.7, 7.2),4.43-4.54 (m, 1H), 4.90 (dt, 1H, J=9.0, 6.2), 5.76 (dd, 1H, J=15.6,1.6), 6.00 (s, 1H), 6.42 (q, 1H, J=0.9), 6.72 (dd, 1H, J=15.6, 5.4),6.86-6.94 (m, 2H), 7.01 (d, 1H, J=9.0), 7.11-7.18 (m, 2H), 7.21 (d, 1H,J=8.7), 7.76 (d, 1H, J=7.2); Anal. (C₃₀H₃₈FN₅O₇) C, H, N.

Example 7 Preparation of Compound A-6:Ethyl-3-[(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-Gln]-E-Propenoate

Preparation of IntermediateEthyl-3-{Boc-L-(4-F)-Phe-L-(Tr-Gln)}-E-Propenoate

This compound was prepared from ethyl-3-(Boc-L-(Tr-Gln))-E-propenoateand Boc-L-(4-F)-Phe-OH using a procedure like that described above(Example 2) for the preparation ofethyl-3-(Boc-L-Phe-L-(Tr-Gln))-E-propenoate: IR (cm⁻¹) 3328, 1707, 1506,1168; ¹H NMR (CDCl₃) δ 1.29 (t, 3H, J=7.2), 1.37 (s, 9H), 1.66-1.78 (m,1H), 1.88-1.98 (m, 1H), 2.32 (t, 2H, J=6.6), 2.85-2.92 (m, 1H),2.97-3.04 (m, 1H), 4.18 (q, 2H, J=7.2), 4.52 (m, 1H), 4.96 (m, 1H), 5.60(d, 1H, J=15.6), 6.56 (d, 1H, J=8.1), 6.66 (dd, 1H, J=15.6, 5.1), 6.80(s, br, 1H), 6.92-6.98 (m, 2H), 7.08-7.12 (m, 2H), 7.17-7.23 (m, 6H),7.24-7.33 (m, 10H); Anal. (C₄₂H₄₆FN₃O₆) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-Val-L-(4-F)-Phe-L-(Tr-Gln)}-E-Propenoate

This compound was prepared fromethyl-3-{Boc-L-(4-F)-Phe-L-(Tr-Gln)}-E-propenoate and Boc-L-Val-OH inthe manner described above (Example 2) for the preparation ofethyl-3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate: IR (cm⁻¹) 3319, 1657,1511, 1172; ¹H NMR (CDCl₃) δ 0.78 (d, 3H, J=6.9), 0.87 (d, 3H, J=6.9),1.29 (t, 3H, J=7.2), 1.37 (s, 9H), 1.69-1.79 (m, 1H), 1.93-2.06 (m, 2H),2.33 (t, 2H, J=7.2), 2.97-3.04 (m, 1H), 3.73-3.77 (m, 1H), 4.18 (q, 2H,J=7.2), 4.42-4.54 (m, 2H), 4.80 (d, 1H, J=6.9), 5.61 (dd, 1H, J=15.6,1.5), 6.44 (d, 1H, J=7.8), 6.69 (dd, 1H, J=15.9, 5.4), 6.71 (s, br, 1H),6.92-6.98 (m, 2H), 7.07-7.13 (m, 2H), 7.18-7.31 (m, 16H), 8.02 (s, 1H);Anal. (C₄₇H₅₅FN₄O₇) C, H, N.

Preparation of IntermediateEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F)-Phe-L-(Tr-Gln)}-E-Propenoate

This compound was prepared fromethyl-3-{Boc-L-Val-L-(4-F)-Phe-L-(Tr-Gln)}-E-propenoate and5-methylisoxazole-3-carbonyl chloride in a manner like that describedabove (Example 2) for the preparation ofethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate:IR (cm⁻¹) 3319, 1657, 1511, 1172; ¹H NMR (CDCl₃) δ 0.84 (d, 3H, J=6.9),0.89 (d, 3H, J=6.9), 1.30 (t, 3H, J=7.2), 1.68-1.80 (m, 1H), 1.95-2.06(m, 1H), 2.08-2.17 (m, 1H), 2.34 (t, 2H, J=7.2), 2.44 (s, 3H), 2.87-2.94(m, 1H), 3.01-3.08 (m, 1H), 4.18 (q, 2H, J=7.2), 4.48-4.56 (m, 2H), 5.68(dd, 1H, J=15.6, 1.8), 6.23 (s, 1H), 6.39 (d, 1H, J=7.8), 6.70 (dd, 1H,J=15.9, 5.4), 6.84-6.90 (m, 3H), 7.04-7.08 (m, 4H), 7.17-7.30 (m, 16H);Anal. (C₄₇H₅₀N₅O₇) C, H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F)-Phe-L-Gln}-E-Propenoate

Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F)-Phe-L-(Tr-Gln)}-E-propenoatewas deprotected using the procedure described above (Example 2) for thepreparation ofethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate:IR (cm⁻¹) 3284, 1652, 1542; ¹H NMR (DMSO-d₆) δ 0.76 (d, 3H, J=6.9), 0.79(d, 3H, J=6.9), 1.20 (t, 3H, J=7.2), 1.57-1.76 (m, 2H), 1.96-2.06 (3H),2.46 (s, 3H), 2.75-2.83 (m, 1H), 2.89-2.96 (m, 1H), 4.09 (q, 2H, J=7.2),4.13-4.25 (m, 1H), 4.35 (m, 1H), 4.49-4.56 (m, 1H), 5.53 (d, 1H,J=15.6), 6.57 (s, 1H), 6.66 (dd, 1H, J=15.6, 5.4), 6.75 (s, br, 1H),6.97-7.03 (m, 2H), 7.17-7.24 (m, 3H), 8.15 (d, 1H, J=7.8), 8.24 (d, 1H,J=8.7), 8.32 (d, 1H, J=8.1); Anal. (C₂₈H₃₆N₅O₇) C, H, N.

Example 8 Preparation of Compound A-7:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

Preparation of Intermediate Boc-L-(4-F-Phe)-Obn

DCC (0.765 g, 3.71 mmol, 1.05 equiv.), benzyl alcohol (0.347 mL, 3.35mmol, 0.95 equiv.) and DMAP (0.022 g, 0.18 mmol, 0.05 equiv.) were addedsequentially to a solution of Boc-L-(4-F-Phe)-OH (1.0 g, 3.53 mmol, 1equiv.) in CH₂Cl₂ (15 mL). After stirring 18 h, the precipitate wasremoved by filtration, and the filtrate was diluted with MTBE (75 mL),washed with 10% KHSO₄ and brine (10 mL each), dried over Na₂SO₄ andevaporated. Purification of the residue by flash column chromatography(12% EtOAc in hexanes) gave Boc-L-(4-F-Phe)-OBn (0.992 g, 79%) as awhite solid. ¹H NMR spectral data matches literature (see Jackson etal., J. Org. Chem. 1992, vol. 57, 3397).

Preparation of Intermediate Boc-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-OBn

Boc-L-(4-F-Phe)-OBn (2.0 g, 5.36 mmol, 1 equiv.) was stirred for 1 h ina mixture of CH₂Cl₂ (20 mL) and TFA (10 mL), then more TFA (10 mL) wasadded and the reaction solution was stirred an additional hour. Thevolatiles were evaporated and the residue was dissolved in DMF (30 mL).Boc-L-α-(t-butyl-Gly)-OH (1.24 g, 5.36 mmol, 1 equiv.) was added, andthe solution was cooled to 0° C. N,N-diisopropylethylamine (2.80 mL,16.1 mmol, 3 equiv.) and HATU (2.04 g, 5.37 mmol, 1 equiv.) were addedsequentially. After stirring 20 min., the reaction mixture was allowedto warm to room temperature over 1 h, then diluted with MTBE (500 mL)and washed with 5% KHSO₄ (100 mL), saturated NaHCO₃ (50 mL), and brine(50 mL). The organic phase was dried and evaporated. Purification of theresidue by flash column chromatography (20% EtOAc in hexanes) gaveBoc-L-α-(t-butyl-Gly)-L-(4-F-Phe)-OBn (2.04 g, 78%) as a white foam:R_(f)=0.49 (25% EtOAc in hexanes); IR (cm⁻¹) 3307, 1737, 1655 br; ¹H NMR(CDCl₃) δ 0.94 (s, 9H), 1.45 (s, 9H), 3.04 (dd, 1H, J=14.2, 5.8), 3.11(dd, 1H, J=14.2, 6.1), 3.79 (d, 1H, J=9.3), 4.88 (dt, 1H, J=7.8, 5.8),5.08 (d, 1H, J=12.0), 5.16-5.23 (m, 1H), 5.19 (d, 1H, J=12.0), 6.08 (d,1H, J=7.8), 6.83-6.97 (m, 4H), 7.28-7.40 (m, 5H); Anal. (C₂₇H₃₅FN₂O₅) C,H, N.

Preparation of IntermediateEthyl-3-{Boc-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 8 mL) was added to a solutionof ethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate(0.309 g, 0.648 mmol, 1 equiv.) in 1,4-dioxane (8 mL). After stirringfor 1.5 h, the volatiles were evaporated to give the crude amine salt asa foam.

Palladium on carbon (10%, 200 mg) was added to a solution ofBoc-L-α-(t-butyl-Gly)-L-(4-F-Phe)-OBn (2.04 g, 4.19 mmol) in EtOAc (200mL). The atmosphere was replaced with hydrogen via balloon. Afterstirring 3 h, the atmosphere was replaced with argon, and the reactionmixture was filtered through #3 and #5 Whatman filter papers. Thefiltrate was evaporated to give a white foam.

This foam was combined with the crude amine salt (prepared above) in DMF(5 mL) and cooled to 0° C. N,N-diisopropylethylamine (0.339 mL, 1.95mmol, 3 equiv.) and HATU (0.247 g, 0.650 mmol, 1 equiv.) were addedsequentially. After stirring 20 min., the reaction mixture was allowedto warm to room temperature over 1 h, then diluted with MTBE (100 mL)and washed with 5% KHSO₄, saturated NaHCO₃ and brine (15 mL each). Theorganic phase was dried and evaporated. Purification of the residue byflash column chromatography (60% EtOAc in hexanes) gaveethyl-3-{Boc-L-α-(t-butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate(0.364 g, 74%) as a white foam: R_(f)=0.34 (60% EtOAc in hexanes); IR(cm⁻¹) 3284, 1713, 1655; ¹H NMR (CDCl₃) δ 1.01 (s, 9H), 1.28 (t, 3H,J=7.2), 1.46 (s, 9H), 1.50-1.63 (m, 211), 1.68-1.81 (m, 1H), 1.85-1.99(m, 1H), 2.09-2.20 (m, 1H), 2.94 (dd, 1H, J=13.5, 5.4), 3.15-3.26 (m,3H), 3.80 (s, 3H), 3.82 (s, 3H), 3.97 (d, 1H, J=9.3), 4.17 (q, 2H,J=7.2), 4.27-4.38 (m, 1H). 4.29 (d, 1H, J=14.3), 4.42 (d, 1H, J=14.3),4.84-4.92 (m, 1H), 5.22 (d, 1H, J=9.6), 5.74 (dd, 1H, J=15.6, 1.6),6.45-6.53 (m, 2H), 6.69 (dd, 1H, J=15.6, 5.4), 6.76-6.87 (m, 3H),7.10-7.28 (m, 3H), 8.26 (d, 1H, J=5.9); Anal. (C₄₀H₅₅FN₄O₉) C, H, N.

Preparation of IntermediateEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-Propenoate

This compound was prepared fromethyl-3-{Boc-L-α-(t-butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoateand isoxazole-5-carbonyl chloride using the procedure described above(Example 2) for the preparation ofethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate:R_(f)=0.60 (10% CH₃OH in CHCl₃); IR (cm⁻¹) 3295, 1713, 1666, 1643; ¹HNMR (CDCl₃) δ 1.07 (s, 9H), 1.29 (t, 3H, J=7.2), 1.51-1.64 (m, 2H),1.71-1.83 (m, 1H), 1.96-2.07 (m, 1H), 2.11-2.21 (m, 1H), 2.49 (s, 3H),2.99 (dd, 1H, J=13.7, 5.9), 3.13-3.26 (m, 3H), 3.80 (s, 3H), 3.81 (s,3H), 4.18 (q, 2H, J=7.2), 4.23-4.48 (m, 4H), 4.85-4.93 (m, 1H), 5.76(dd, 1H, J=15.6, 1.4), 6.40-6.52 (m, 3H), 6.71 (dd, 1H, J=15.6, 5.3),6.79-6.88 (m, 2H, 6.92 (d, 1H, J=9.0), 7.09-7.22 (m, 3H), 7.37 (d, 1H,J=9.0), 8.27 (d, 1H, J=6.2); Anal. (C₄₀H₅₀FN₅O₉.0.25H₂O) C, H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

This compound was prepared fromethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-α-(t-Butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoateusing a procedure as described above (Example 6) for the preparation ofethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-propenoate:R_(f)=0.30 (5% CH₃OH in CH₂Cl₂); IR (cm⁻¹) 3307 br, 1684, 1660; ¹H NMR(CDCl₃) δ 1.02 (s, 9H), 1.29 (t, 3H, J=7.2), 1.51-1.61 (m, 1H),1.75-1.97 (m, 2H), 2.14-2.25 (m, 1H), 2.28-2.40 (m, 1H), 2.50 (s, 3H),3.03 (d, 2H, J=6.5), 3.27-3.42 (m, 2H), 4.18 (q, 2H, J=7.2), 4.36 (d,1H, J=9.8), 4.52-4.63 (m, 1H), 4.86-4.95 (m, 1H), 5.71 (dd, 1H, J=15.6,1.4), 6.44 (s, 1H), 6.57-6.64 (m, 1H), 6.73 (dd, 1H, J=15.6, 5.3),6.83-6.91 (m, 2H), 7.09-7.15 (m, 2H), 7.28-7.36 (m, 2H), 7.59 (d, 1H,J=8.1); Anal. (C₃₁H₄₀FN₅O₇) C, H, N.

Example 9 Preparation of Compound A-8:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Piper-Ala)}-E-Propenoate

Preparation of Intermediate(2′S,4S,4″S)-4-{2′-(4-Benzyl-2″-oxo-oxazolidine-3″-carbonyl)-5′-hydroxypentyl}-2-dimethyloxazolidine-3-carboxylicAcid tert-Butyl Ester

A solution of borane-tetrahydrofuran complex (0.96 mL of a 1.0 Msolution in TBF, 0.96 mmol, 1 equiv.) was added to a 0° C. solution of(2′S,4S,4″S)-4-{2′-(4″-benzyl-2′-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (prepared as described in Example 6, 0.455 g,0.963 mmol, 1 equiv.) in THF (3 mL). After stirring 30 min., water (3mL) and sodium perborate tetrahydrate (0.148 g, 0.962 mmol, 1 equiv.)were added, and the ice bath was removed. After an additional hour, thereaction mixture was diluted with MTBE (125 mL), washed with water (15mL) and brine (2×15 mL), dried over Na₂SO₄, and concentrated.Purification of the residue by flash column chromatography (50% EtOAc inhexanes) provided(2′S,4S,4″S)-4-{2′-(4″-benzyl-2″-oxo-oxazolidine-3″-carbonyl)-5′-hydroxypentyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (0.339 g, 72%) as a colorless glass: R_(f)=0.41(50% EtOAc in hexanes); IR (cm⁻¹) 3486, 1780, 1693; ¹H NMR (CDCl₃) δ1.42-1.85 (m, 21H), 2.13-2.24 (m, 1H), 2.70 (dd, 1H, J=13.1, 10.0),3.29-3.38 (m, 1H), 3.61-4.22 (m, 8H), 4.63-4.76 (m, 1H), 7.19-7.38 (m,5H); Anal. (C₂₆H₃₈N₂O₇.0.5H₂O) C, H, N.

Preparation of Intermediate(1S,3′S)-{2-(1′-(2″,4″-Dimethoxybenzyl)-2′-oxo-piperidin-3′-yl)-1-hydroxymethylethyl}-carbamicAcid tert-Butyl Ester

(2′S,4S,4″S)-4-{2′-(4″-Benzyl-2″-oxo-oxazolidine-3″-carbonyl)-5′-hydroxypentyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (3.88 g, 7.92 mmol, 1 equiv.) was dissolved inEt₃N (3.97 mL, 28.51 mmol, 3.6 equiv.). The mixture was cooled to −12°C., and a solution of sulfur trioxide-pyridine complex (5.04 g, 31.67mmol, 4 equiv.) in DMSO (150 mL) was added at a rate to maintain thetemperature between 8-17° C. The solution was stirred at 23° C. for 3 h.The reaction mixture was cooled in an ice water bath and quenched by theaddition of H₂O (150 mL). The resulting solution was extracted withEtOAc (2×150 mL). The combined organic layers were washed with 5% citricacid (100 mL), brine (100 mL), dried over Na₂SO₄ and filtered. Thesolvent was removed under reduced pressure and the residue was driedunder vacuum to give a white foam (3.53 g).

To a solution of this material (3.53 g, 7.22 mmol, 1 equiv.) in a 2:1mixture of THF and EtOH (120 mL) was added 2,4-dimethoxybenzylaminehydrochloride (5.88 g, 28.89 mmol, 4 equiv.), NaOAc (2.37 g, 28.89 mmol,4 equiv.), and NaBH₃CN (0.908 g, 14.45 mmol, 2 equiv.). The reactionmixture was stirred overnight (20 h) and then diluted with MTBE (200mL). The organic layer was washed with 10% KHSO₄ (100 mL), saturatedNaHCO₃ (100 mL), and brine (100 mL), and dried over Na₂SO₄, andconcentrated to give a pale yellow foam.

To a solution of this foam (3.34 g, 7.22 mmol, 1 equiv.) in CH₃OH (50mL) was added p-toluenesulfonic acid (0.275 g, 1.44 mmol, 0.2 equiv.).The reaction mixture was stirred at 50° C. for 2.5 h and then wasdiluted with CH₂Cl₂ (100 mL). The organic layer was washed withsaturated NaHCO₃ (100 mL), dried over Na₂SO₄, and concentrated. Theresidue was purified by flash column chromatography (3% CH₃OH in CH₂Cl₂)to give(1S,3′S)-{2-(1′-(2″,4″-dimethoxybenzyl)-2′-oxo-piperidin-3′-yl)-1-hydroxymethyl-ethyl}-carbamicacid tert-butyl ester as a white foam (1.33 g, 44% over three steps): ¹HNMR (CDCl₃): δ 1.44 (s, 9H), 1.71-1.85 (m, 2H), 1.92-1.98 (m, 2H),2.40-2.48 (m, 1H), 2.71-2.78 (m, 1H), 3.19-3.32 (m, 2H), 3.45-3.69 (m,4H), 4.11-4.20 (m, 2H), 4.68 (m, 1H), 5.47 (m, 1H), 6.44 (s, 1H),7.20-7.33 (m, 2H).

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Piper-Ala)}-E-Propenoate

(1S,3′S)-{2-(1′-(2″,4″-Dimethoxybenzyl)-2′-oxo-piperidin-3′-yl)-1-hydroxy-methylethyl}-carbamicacid tert-butyl ester was converted to the productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Piper-Ala)}-E-propenoatein a manner analogous to the conversion of(1S,3′S)-{2-(1′-(2″,4″-dimethoxybenzyl)-2′-oxo-piperidin-3′-yl]-1-hydroxymethylethyl)-carbamicacid tert-butyl ester to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-propenoatedescribed in Example 6 above: R_(f)=0.24 (5% CH₃OH in CH₂Cl₂); IR (cm⁻¹)3284 br, 1713, 1655, 1637 br; ¹H NMR (CDCl₃) δ 0.94 (d, 3H, J=6.8), 0.98(d, 3H, J=6.8), 1.29 (t, 3H, J=7.2), 1.43-1.56 (m, 2H), 1.66-1.78 (m,1H), 1.83-2.05 (m, 4H), 2.16-2.28 (m, 1H), 2.49 (s, 3H), 3.00 (dd, 1H,J=13.7, 6.2), 3.13 (dd, 1H, J=13.7, 5.9), 3.21-3.37 (m, 2H), 4.18 (q,2H, J=7.2), 4.36-4.45 (m, 2H), 4.80-4.88 (m, 1H), 5.76 (dd, 1H, J=15.6,1.6), 5.96 (s, 1H), 6.43 (s, 1H), 6.70 (dd, 1H, J=15.6, 5.3), 6.81 (d,1H, J=8.7), 6.86-6.98 (m, 2H), 7.09-7.19 (m, 2H), 7.22-7.29 (m, 1H),8.07 (d, 1H, J=6.5).

Example 10 Preparation of Compound B-1:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-Gln}-E-Propenoate

Preparation of Intermediate trans-6-Methyl-hept-4-enoic Acid

A solution of isobutyraldehyde (9.59 g, 133 mmol, 1 equiv.) in THF (50mL) was added dropwise via addition funnel to a solution ofvinylmagnesium bromide (133 mL of a 1.0 M solution in THF, 133 mmol, 1.0equiv.) in THF (300 mL) at 0° C. Upon completion of the addition, thereaction mixture was stirred for 30 min. at 0° C., and then ethylmalonyl chloride (17.0 mL, 133 mmol, 1.0 equiv.) was added. Afterstirring for 1 h at 0° C., the reaction mixture was partitioned betweensaturated NH₄Cl (150 mL) and a 1:1 mixture of EtOAc and hexanes (2×200mL). The combined organic layers were dried over Na₂SO₄ and wereconcentrated. Purification of the residue by filtration through silicagel (eluting with 5% EtOAc in hexanes) afforded the intermediatemalonate ester (11.5 g, 40% yield). This material was not characterized,but was combined (neat) with Ti(OEt)₄ (1.13 mL, 5.39 mmol, 0.10 equiv.)and was heated to 190° C. for 4 h, and then was cooled to 60° C. EtOH(50 mL) and 6.0 M KOH (50 mL) were added sequentially, and the brownreaction mixture was refluxed for 4 h. After cooling to 23° C., thereaction mixture was filtered through a medium frit, and the filtratewas partitioned between water (150 mL) and Et₂O (2×150 mL). The aqueouslayer was then acidified to pH=2 (as indicated by pH paper) withconcentrated HCl and was extracted with a 1:1 mixture of EtOAc andhexanes (2×150 mL). The combined organic layers were dried over Na₂SO₄,concentrated, and the residue was distilled at reduced pressure toafford trans-6-methyl-hept-4-enoic acid (3.58 g, 47%) as a colorlessliquid: bp 107-112° C. (1 Torr); IR (cm⁻¹) 2960, 1711; ¹H NMR (CDCl₃) δ0.96 (d, 6H, J=6.5), 2.18-2.45 (m, 5H), 5.31-5.50 (m, 2H); Anal.(C₈H₁₄O₂) C, H.

Preparation of Intermediate trans-6-Methyl-hept-4-enoic Acid(2R-hydroxy-1R-methyl-2-phenyl-ethyl)-methyl Amide

Oxalyl chloride (2.25 mL, 25.8 mmol, 1.05 equiv.) was added to asolution of trans-6-methyl-hept-4-enoic acid (3.50 g, 24.6 mmol, 1equiv.) and N,N-dimethylformamide (0.03 mL, 0.39 mmol, 0.016 equiv.) inbenzene (60 mL) at 23° C. The reaction mixture was stirred at 23° C. for2 h, and then was concentrated under reduced pressure. The resulting oilwas dissolved in THF (20 mL) and was added via cannula to a solution of(1R,2R)-(−)-pseudoephedrine (3.87 g, 23.4 mmol, 1 equiv.) andtriethylamine (3.92 mL, 28.1 mmol, 1.2 equiv.) in THF (150 mL) at 0° C.The reaction mixture was stirred at 0° C. for 30 min., then waspartitioned between half-saturated NH₄Cl (150 mL) and EtOAc (2×150 mL).The combined organic layers were dried over Na₂SO₄, concentrated, andthe residue purified by flash column chromatography (gradient elution40→50% EtOAc in hexanes) to afford trans-6-methyl-hept-4-enoic acid(2R-hydroxy-1R-methyl-2-phenyl-ethyl)-methyl amide (6.31 g, 93%) as aviscous oil: R_(f)=0.35 (50% EtOAc in hexanes); IR (cm⁻¹) 3382, 1622; ¹HNMR (CDCl₃, mixture of rotamers) δ 0.96 (d, J=6.8), 0.97 (d, J=6.5),1.11 (d, J=6.9), 2.18-2.59 (m), 2.82 (s), 2.92 (s), 3.99-4.04 (m),4.32-4.42 (m), 4.44-4.49 (m), 4.55-4.62 (m), 5.32-5.49 (m), 7.24-7.42(m); Anal. (C₁₈H₂₇NO₂) C, H, N.

Preparation of Intermediatetrans-6-Methyl-2S-(4-fluorobenzyl)-hept-4-enoic Acid(2R-Hydroxy-1R-methyl-2-phenylethyl)methyl Amide

n-Butyllithium (32.5 mL of a 1.6 M solution in hexanes, 52.0 mmol, 3.1equiv.) was added to a suspension of anhydrous lithium chloride (7.18 g,169 mmol, 10 equiv.) and diisopropylamine (7.80 mL, 55.7 mmol, 3.3equiv.) in THF (250 mL) at −78° C. The reaction mixture was stirred for30 min. at −78° C., was maintained at 0° C. for 5 min., and subsequentlycooled again to −78° C. trans-6-Methyl-hept-4-enoic acid(2R-hydroxy-1R-methyl-2-phenyl-ethyl)-methyl amide (4.91 g, 17.0 mmol, 1equiv) in THF (50 mL) was added via cannula, and the resulting solutionwas stirred at −78° C. for 1.75 h, maintained at 0° C. for 20 min.,stirred at 23° C. for 5 min., and then was cooled again to 0° C. Asolution of 4-fluorobenzyl bromide (6.34 mL, 50.9 mmol, 3 equiv.) in THF(15 mL) was added and the reaction mixture was stirred at 0° C. for 30min., which was then partitioned between half-saturated NH₄Cl (230 mL)and a 1:1 mixture of EtOAc and hexanes (200 mL, 2×150 mL). The combinedorganic layers were dried over Na₂SO₄ and were concentrated.Purification of the residue by flash column chromatography (gradientelution 20→40% EtOAc in hexanes) providedtrans-6-methyl-2S-(4-fluorobenzyl)-hept-4-enoic acid(2R-hydroxy-1R-methyl-2-phenylethyl)methyl amide (6.33 g, 94%) as aviscous oil: R_(f)=0.38 (40% EtOAc in hexanes); IR (cm⁻¹) 3378, 1614; ¹HNMR (CDCl₃, mixture of rotamers) δ 0.85-0.95 (m), 0.96 (d, J=6.8),2.10-2.32 (m), 2.34-2.46 (m), 2.58 (s), 2.67-2.79 (m), 2.82-2.94 (m),3.00-3.18 (m), 3.94 (br), 4.37-4.52 (m), 5.24-5.42 (m), 5.44-5.56 (m),6.89-7.01 (m), 7.08-7.14 (m), 7.19-7.38 (m); Anal. (C₂₅H₃₂FNO₂) C, H, N.

Preparation of Intermediate5S-(1R-Bromo-2-methylpropyl)-3R-(4-fluorobenzyl)dihydrofuran-2-one

N-Bromosuccinimide (2.93 g, 16.5 mmol, 1.05 equiv.) was added in smallportions over 10 minutes to a solution oftrans-6-methyl-2S-(4-fluorobenzyl)-hept-4-enoic acid(2R-hydroxy-1R-methyl-2-phenylethyl)methyl amide (6.24 g, 15.7 mmol, 1equiv.) and glacial acetic acid (4.49 mL, 78.4 mmol, 5 equiv.) in a 4:1mixture of THF and H₂O (165 mL) at 0° C. The resulting yellow solutionwas stirred for 15 min. at 0° C., and then was warmed to 23° C. andsubsequently refluxed for 45 min. After cooling to 23° C., the reactionmixture was partitioned between half-saturated NaHCO₃ (200 mL) and a 1:1mixture of EtOAc and hexanes (2×200 mL, 100 mL). The combined organiclayers were dried over Na₂SO₄ and were concentrated. Flashchromatographic purification of the residue (gradient elution 4→10%EtOAc in hexanes) gave5S-(1R-bromo-2-methylpropyl)-3R-(4-fluorobenzyl)dihydrofuran-2-one (4.14g, 80%) as a pale yellow oil (containing approximately 5-10%unidentified impurities by ¹H NMR): R_(f)=0.56 (25% EtOAc in hexanes);IR (cm⁻¹) 1772; ¹H NMR (CDCl₃, major isomer) δ 0.94 (d, 3H, J=6.5), 1.00(d, 3H, J=6.8), 2.05-2.35 (m, 3H), 2.83 (dd, 1H, J=13.6, 8.4), 2.92-3.03(m, 1H), 3.11 (dd, 1H, J=13.6, 4.7), 3.90 (dd, 1H, J=9.0, 3.7),4.33-4.40 (m, 1H), 6.98-7.06 (m, 2H), 7.14-7.20 (m, 2H); Anal.(C₁₅H₁₈BrFO₂) C, H.

Preparation of Intermediate5S-(1S-Azido-2-methylpropyl)-3R-(4-fluorobenzyl)dihydrofuran-2-one

A suspension of sodium azide (1.90 g, 29.2 mmol, 2.5 equiv.) and5S-(1R-bromo-2-methylpropyl)-3R-(4-fluorobenzyl)dihydrofuran-2-one (3.85g, 11.7 mmol, 1 equiv.) in N,N-dimethylformamide (40 mL) was heated at50° C. for 67 hours. The reaction mixture was cooled to 23° C. and waspartitioned between half-saturated NaCl (200 mL) and a 1:1:1 mixture ofEtOAc, hexanes and acetone (2×200 mL, 100 mL). The combined organiclayers were dried over Na₂SO₄, concentrated, and the residue purified byflash column chromatography (gradient elution 9→17% EtOAc in hexanes) togive 5S-(1S-azido-2-methylpropyl)-3R-(4-fluorobenzyl)dihydrofuran-2-one(2.10 g, 62%) as a white solid (containing approximately 5-10%unidentified impurities by ¹H NMR): mp 91-96° C.; R_(f)=0.44 (25% EtOAcin hexanes); IR (cm⁻¹) 2097, 1772; ¹H NMR (CDCl₃, major isomer) δ 0.99(d, 3H, J 6.5), 1.02 (d, 3H, J=6.8), 1.95-2.20 (m, 3H), 2.78-2.88 (m,1H), 2.94 (dd, 1H, J=7.0, 4.2), 3.03-3.17 (m, 2H), 4.37-4.43 (m, 1H),6.97-7.09 (m, 2H), 7.14-7.21 (m, 2H).

Preparation of Intermediate{2-Methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamicAcid tert-Butyl Ester

A suspension of5S-(1S-azido-2-methylpropyl)-3R-(4-fluorobenzyl)dihydrofuran-2-one (2.02g, 6.93 mmol, 1 equiv.), di-tert-butyl dicarbonate (2.12 g, 9.71 mmol,1.4 equiv.) and Pd/C (10%, 0.20 g) in CH₃OH (100 mL) was stirred under ahydrogen atmosphere (balloon) for 16 hours. The reaction mixture wasvacuum filtered through Whatman #3 paper and concentrated. Purificationof the residue by flash column chromatography (15% EtOAc in hexanes)provided{2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamicacid tert-butyl ester (1.58 g, 62%) as a white foam: R_(f)=0.80 (5% MeOHin CH₂Cl₂); IR (cm⁻¹) 3331, 1766, 1702; ¹H NMR (CDCl₃) δ 0.93 (d, 3H,J=6.8), 0.95 (d, 3H, J=6.5), 1.41 (s, 9H), 1.71-1.83 (m, 1H), 1.95-2.06(m, 1H), 2.16-2.27 (m, 1H), 2.80 (dd, 1H, J=13.5, 8.6), 2.88-2.99 (m,1H), 3.09 (dd, 1H, J=13.5, 4.4), 3.32-3.40 (m, 1H), 4.42-4.48 (m, 2H),6.95-7.03 (m, 2H), 7.11-7.18 (m, 2H); Anal. (C₂₀H₂₈FNO₄) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-(Tr-Gln)}-E-Propenoate

Lithium hydroxide (9.62 mL of a 1 M aqueous solution, 9.62 mmol, 5equiv.) was added to a solution of{2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamicacid tert-butyl ester (0.703 g, 1.92 mmol, 1 equiv.) in DME (25 mL) at23° C. The resulting suspension was stirred at 23° C. for 30 min., andthen was partitioned between 10% KHSO₄ (50 mL) and CH₂Cl₂ (3×100 mL).The combined organic layers were dried over Na₂SO₄, concentrated, andthe residue dissolved in CH₂Cl₂ (30 mL). Powdered 4 Å molecular sieves(0.70 g), 4-methylmorpholine N-oxide (0.451 g, 3.85 mmol, 2 equiv.), andtetrapropylammonium perruthenate (0.068 g, 0.19 mmol, 0.10 equiv.) wereadded sequentially. The resulting dark reaction mixture was stirred for1.33 hours at 23° C., then was vacuum filtered through Whatman #3 paperand then through Whatman #5 paper. The filtrate was concentrated underreduced pressure to provide a dark residue, which was dissolved inCH₂Cl₂ (30 mL). Crude ethyl-3-(H₂N-L-(Tr-Gln))-E-propenoate·HCl (2.30mmol, 1.2 equiv., prepared as described in Example 2 for the preparationofethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate),4-methylmorpholine (0.846 mL, 7.69 mmol, 4 equiv.), HOBt (0.390 g, 2.89mmol, 1.5 equiv.), and EDC (0.553 g, 2.88 mmol, 1.5 equiv.) were addedsequentially, and the reaction mixture was stirred for 19 hours at 23°C. and then was partitioned between brine (100 mL) and CH₂Cl₂ (3×100mL). The combined organic layers were dried over Na₂SO₄ and wereconcentrated. Purification of the residue by flash column chromatography(gradient elution 35→40% EtOAc in hexanes) providedethyl-3-{Boc-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (0.820g, 53%) as a tan foam: R_(f)=0.50 (50% EtOAc in hexanes); IR (cm⁻¹)3307, 1708, 1666; ¹H NMR (CDCl₃) δ 0.67 (d, 3H, J=6.8), 0.92 (d, 3H,J=6.8), 1.28 (t, 3H, J=7.2), 1.40 (s, 9H), 1.53-1.67 (m, 1H), 1.91-2.04(m, 2H), 2.32-2.41 (m, 2H), 2.46-2.55 (m, 1H), 2.63 (dd, 1H, J=12.1,5.9), 2.69-2.80 (m, 1H), 2.83 (dd, 1H, J=12.1, 8.2), 3.03 (dd, 1H,J=17.7, 10.0), 4.05-4.11 (m, 1H), 4.17 (q, 2H, J=7.2), 4.40-4.50 (m,1H), 4.84 (d, 1H, J=8.4), 5.38 (d, 1H, J=15.7), 6.01 (d, 1H, J=8.4),6.60 (dd, 1H, J=15.7, 5.0), 6.92-6.99 (m, 2H), 7.03-7.12 (m, 3H),7.17-7.30 (m, 15H); Anal. (C₄₈H₅₆FN₃O₇) C, H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-Gln)}-E-Propenoate

Ethyl-3-{Boc-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate wasconverted to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-Gln}-E-propenoatein a manner analogous to that described in Example 2 above for theconversion of ethyl-3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate toproductethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate:mp=220° C. (dec); R_(f)=0.35 (10% CH₃OH in CH₂Cl₂); IR (cm⁻¹) 3277,1715, 1643; ¹H NMR (DMSO-d₆) δ 0.81 (d, 3H, J=6.2), 0.87 (d, 3H, J=6.9),1.21 (t, 3H, J=6.5), 1.59-1.67 (m, 2H), 2.03 (s, br, 2H), 2.21-2.24 (m,1H), 2.46 (s, 3H), 2.57-2.68 (m, 3H), 2.80-2.95 (m, 2H), 4.09 (q, 2H,J=6.5), 4.30-4.34 (m, 2H), 5.41 (d, 1H, J=15.5), 6.55 (s, 1H), 6.61 (dd,1H, J=15.5, 5.5), 6.73 (s, 1H), 6.99-7.16 (m, 5H), 8.01 (d, 1H, J=7.8),8.69 (d, 1H, J=8.7); Anal. (C₂₉H₃₇FN₄O₇) C, H, N.

Example 11 Preparation of Compound A-10:Ethyl-3-((5′-Methylisoxazole-3′-carbonyl)-L-Leu-NCH₃-L-Phe-L-Gln}-E-Propenoate

Preparation of Intermediate Boc-L-Leu-NCH₃-L-Phe-OCH₃

NCH₃-L-Phe-OCH₃.HCl (1.4 g) was dissolved in CH₂Cl₂ (50 mL) and pouredinto a combination of aqueous (aq) 1 N NaOH (7 mL) and saturated aqueousNaHCO₃ (25 mL). After mixing, the organic phase was separated and theaqueous phase was washed with CH₂Cl₂ (3×50 mL). The combined organicphases were dried over Na₂SO₄ and evaporated to give the free amine as aclear colorless oil (1.14 g, 5.90 mmol). A solution of this amine and(iPr)₂NEt (1.13 mL, 6.49 mmol) in DMF (10 mL) was added dropwise to a 0°C. solution of Boc-L-Leu-OH (1.50 g, 6.49 mmol) and HOBt (0.877 g, 6.49mmol) in DMF (10 mL). DCC (1.47 g, 7.12 mmol) was added. The reactionmixture was stirred at 0° C. for 1 h, and was then stirred at 23° C. for48 h. The mixture was filtered to remove the precipitate (ppt) and thefiltrate was evaporated. The residue was dissolved in CH₂Cl₂ (200 mL),washed with saturated aqueous NaHCO₃ (40 mL), dried over Na₂SO₄ andevaporated. The residue was purified by flash column chromatography (25%EtOAc in hexanes) to give Boc-L-Leu-NCH₃-L-Phe-OCH₃ as a white solid(2.04 g, 85%): mp=126-127° C.; IR (cm⁻) 3401, 3319, 1743, 1708, 1649; ¹HNMR (CDCl₃) (major isomer) δ 0.92 (d, 3H, J=6.8), 0.95 (d, 3H, J=6.5),1.32-1.48 (m, 2H), 1.41 (s, 9H), 1.61-1.77 (m, 1H), 2.90 (s, 3H), 3.04(dd, 1H, J=14.5, 10.5), 3.37 (dd, 1H, J=14.5, 5.5), 3.72 (s, 3H),4.48-4.57 (m, 1H), 4.98-5.04 (m, 1H), 5.20 (dd, 1H, J=10.5, 5.5),7.16-7.32 (m 5H); Anal. (C₂₂H₃₄N₂O₅) C, H, N.

Preparation of Intermediate Boc-L-Leu-NCH₃-L-Phe-OH

Boc-L-Leu-NCH₃-L-Phe-OCH₃ (0.625 g, 1.54 mmol) was dissolved in CH₃OH(20 mL) and cooled to 0° C. Aqueous NaOH (6.15 mL of a 2N solution, 12.3mmol) was added dropwise. The reaction mixture was stirred for 3 h at23° C., and then poured into 10% aqueous KHSO₄ (150 mL). This mixturewas extracted with CH₂Cl₂ (3×100 mL), and the combined organic phaseswere dried over Na₂SO₄ and evaporated to give Boc-L-Leu-NCH₃-L-Phe-OH asa white foam (0.617 g, quant.), which was used without purification.

Preparation of IntermediateEthyl-3-{BOC-L-Leu-NCH₃-L-Phe-L-(Tr-Gln)}-E-Propenoate

This intermediate was prepared from Boc-L-Leu-NCH₃-L-Phe-OH andethyl-3-{H₂N-L-(Tr-Gln)}-E-propenoate.HCl (prepared as described inExample 2) in a manner analogous to that described for the preparationof ethyl-3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate in Example 2 above:IR (cm⁻¹) 3295, 1713, 1672, 1649; ¹H NMR (CDCl₃) (mixture of isomers) δ0.65 (d, J=6.2), 0.66 (d, J=6.5), 0.84 (d, J=6.5), 0.88 (d, J=6.5),1.02-1.22 (m), 1.23-1.38 (m), 1.33 (s), 1.41 (s), 1.55-1.82 (m),1.89-2.07 (m), 2.23-2.30 (m), 2.90 (s), 2.94 (s), 3.01 (dd, J=14.6,10.9), 3.03-3.13 (m), 3.26-3.37 (m), 3.27 (dd, J=14.6, 3.4), 3.42-3.54(m), 4.00-4.22 (m), 4.37-4.73 (m), 4.82-4.89 (m), 5.53-5.70 (m), 5.95(dd, J=15.9, 1.2), 6.23-6.28 (m), 6.66-6.75 (m), 6.79-6.89 (m),7.09-7.34 (m), 8.14 (d, J=8.7); Anal. (C₄₉H₆₀N₄O₇) C, H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Leu-NCH₃-L-Phe-L-Gln}-E-Propenoate

Ethyl-3-{Boc-L-Leu-NCH₃-L-Phe-L-(Tr-Gln)}-E-propenoate was converted toproductethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-NCH₃-L-Phe-L-Gln}-E-propenoatein a manner analogous to that described in Example 2 above for theconversion of ethyl-3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate toproductethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate:R_(f)=0.23 (5% CH₃OH in CH₂Cl₂); IR (cm⁻) 3295, 1713, 1666, 1637; ¹H NMR(CDCl₃) (mixture of isomers) δ 0.65 (d, J=6.5), 0.71 (d, J=6.5), 0.93(d, J=6.5), 0.94 (d, J=6.5), 1.30 (t, J=7.2), 1.24-1.73 (m), 1.81-2.22(m), 2.45 (s), 2.48 (s), 2.86-2.93 (m), 2.96 (s), 2.97 (s), 3.03-3.14(m), 3.21-3.31 (m), 3.48 (dd, J=14.0, 5.9), 4.19 (q, J=7.2), 4.20 (q,J=7.2), 4.38-4.45 (m), 4.52-4.70 (m), 4.74-4.81 (m), 5.62-5.67 (m),5.73-5.79 (m), 5.81 (dd, J=15.6, 1.6), 5.99 (dd, J=15.6, 1.6), 6.03-6.09(m), 6.35 (s), 6.39 (s), 6.40-6.45 (m), 6.77-6.94 (m), 7.42 (d, J=7.2),8.13 (d, J=7.8).

Example 12 Preparation of Compound C-1:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-Gln}-E-Propenoate

Preparation of Intermediate Allyl(S)-1-Hydroxy-3-(4-fluorophenyl)propionate

In a flask fitted with a thermometer and a reflux condenser wasdissolved methyl (S)-1-hydroxy-3-(4-fluorophenyl)propionate (preparedfrom L-H₂N-(4-F-Phe)-OCH₃ by the method described in Hoffman et al.,Tetrahedron 1992, vol. 48, 3007) (0.99 g, 5.0 mmol) in allyl alcohol (50mL). Titanium tetraisopropoxide (1.53 mL, 5.0 mmol) was if added, andthe reaction brought to 90° C. for 3.5 h. The reaction was cooled toroom temperature and poured into 250 mL of 1:1 EtOAc/saturated NH₄Clsolution. The organic phase was separated and washed with water (100mL), brine (100 mL), dried (MgSO₄), and the solvent removed. The residuewas subjected to flash column chromatography eluting with a gradient of5-10% EtOAc/hexanes to afford 0.77 g (68%) of allyl(S)-1-hydroxy-3-(4-fluorophenyl)propionate as a clear liquid: R_(f)=0.21(15% EtOAc/hexanes); IR (neat) 3470 (broad), 1734, 1510, 1221 cm⁻¹; ¹HNMR (DMSO-d₆) δ 1.24-1.27 (m, 1H), 2.92-2.99 (m, 1H), 3.09-3.15 (m, 1H),4.43-4.47 (m, 1H), 4.65 (d, 2H, J=5.9), 5.28-5.37 (m, 2H), 5.86-5.95 (m,1H), 6.95-7.01 (m, 2H), 7.16-7.21 (m, 2H).

Preparation of Intermediate Boc-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂

Allyl (S)-1-hydroxy-3-(4-fluorophenyl)propionate (0.070 g, 0.31 mmol)was dissolved in CH₂Cl₂ (20 mL). Boc-L-Val-OH (0.068 g, 0.31 mmol) wasadded, followed by DMAP (0.004 g, 0.03 mmol) and DCC (0.067 g, 0.33mmol). The reaction was stirred at room temperature overnight, and thesolvent was removed in vacuo. The residue was subjected to flash columnchromatography eluting with a gradient of 3-5% EtOAc/hexanes. TheBoc-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂ product was obtained as 0.12 g (90%)of a clear oil: R_(f)=0.18 (10% EtOAc/hexanes); IR (neat) 1752, 17171510 cm⁻¹; ¹H NMR (DMSO-d₆) δ 0.80-0.85 (m, 6H), 1.36 (s, 9H), 1.97-2.04(m, 1H), 3.07-3.15 (m, 2H), 3.89-3.94 (m, 1H), 4.51-4.55 (m, 2H),5.17-5.30 (m, 3H), 5.75-5.84 (m, 1H), 7.06-7.17 (m, 3H), 7.27-7.32 (m,2H); Anal. (C₂₂H₃₀NO₆F) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-Val-O-L-(4-F-Phe)-L-(Tr-Gln)}-E-Propenoate

Boc-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂ (0.65 g, 1.52 mmol) was dissolved inTHF (15 mL). Tetrakis(triphenylphosphine)palladium(0) (0.035 g, 0.03mmol) was added, and the reaction stirred 5 min. at 23° C. Morpholine(0.16 mL, 1.83 mmol) was added dropwise, and the reaction stirred atroom temperature for 2 h. The solvent was removed in vacuo, and theresidue taken up in 50 mL of 4:1 hexanes/Et₂O. The product was extractedinto sat. NaHCO₃ solution (50 mL), and the organic phase discarded. Theaqueous phase was acidified to pH=1-2 with solid KHSO₄, and the productre-extracted into EtOAc (50 mL). The organic phase was washed with brine(50 mL), dried (MgSO₄), and concentrated to give 0.50 g (86%) of thefree acid as a clear oil. This material was dissolved in DMF (6 mL).Diisopropylethylamine (0.43 mL, 2.50 mmol) was added, followed byethyl-3-{H₂N-L-(Tr-Gln)}-E-propenoate.HCl (prepared as described inExample 2 above, 0.55 g, 1.25 mmol). The reaction was cooled to 0° C.HATU (0.48 g, 1.25 mmol) was added, and the reaction allowed to warm toroom temperature. The DMF was removed in vacuo. The residue wasdissolved with EtOAc (30 mL), and the organic phase washed consecutivelywith 10% HCl solution (25 mL), saturated NaHCO₃ solution (25 mL), H₂O(25 mL), and brine (25 mL). The organic layer was dried (MgSO₄),filtered, and concentrated, and the residue was purified by flash columnchromatography (0→1.0% MeOH/CH₂Cl₂) to give 0.40 g (39%) ofethyl-3-{Boc-L-Val-O-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate as a whiteamorphous solid: R_(f)=0.25 (3% MeOH/CHCl₃); IR(KBr) 1691, 1512, 1159cm⁻¹; ¹H NMR (DMSO-d₆) δ 0.78-0.83 (m, 6H), 1.20 (t, 3H, J=7.0), 1.35(s, 9H), 1.58-1.66 (m, 2H), 1.96-2.02 (m, 1H), 2.19-2.33 (m, 2H),2.99-3.02 (m, 2H), 3.82-3.87 (m, 1H), 4.09 (q, 2H, J=7.0), 4.33-4.37 (m,1H), 5.03-5.08 (m, 1H), 5.60 (d, 1H, J=15.8), 6.67 (dd, 1H, J=15.8,5.5), 7.02-7.08 (m, 2H), 7.14-7.28 (m, 18H), 8.12 (d, 1H, J=8.1), 8.59(s, 1H); Anal. (C₄₇H₅₄N₃O₈F) C, H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-Gln}-E-Propenoate

Ethyl-3-{Boc-L-Val-O-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate was convertedto productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-Gln}-E-propenoatein a manner analogous to that described in Example 2 above for theconversion of ethyl-3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate toproductethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate:R_(f)=0.05 (3% MeOH/CHCl₃); IR (KBr) 1746, 1719, 1661, 1549 cm⁻¹; ¹H NMR(DMSO-d₆) δ 0.87 (d, 3H, J=6.6), 0.92 (d, 3H, J=6.6), 1.20 (t, 3H,J=7.0), 1.61-1.74 (m, 2H), 1.96-2.01 (m, 2H), 2.15-2.22 (m, 1H), 2.46(s, 3H), 3.00-3.03 (m, 2H), 4.10 (q, 2H, J=7.0), 4.27-4.32 (m, 1H),4.33-4.38 (m, 1H), 5.06-5.11 (m, 1H), 5.63 (d, 1H, J=15.6), 6.54 (s,1H), 6.68 (dd, 1H, J=15.6, 5.5), 6.78 (s, br, 1H), 6.95-7.00 (m, 2H),7.20-7.24 (m, 3H), 8.06 (d, 1H, J=8.1), 8.87 (d, 1H, J=7.7); Anal.(C₂₈H₃₅N₄O₈F) C, H, N.

Example 13 Preparation of Compound A-11:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-Propenoate

Preparation of IntermediateEthyl-3-{Boc-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-Propenoate

Boc-(4-F-Phe)-OH (4.46 g, 15.75 mmol, 1 equiv.) and CH₃I (7.84 mL, 126mmol, 8 equiv.) were dissolved in dry THF (100 mL) and cooled to 0° C.NaH (1.89 g, 47.25 mmol, 3 equiv.) was added to this solution withvigorous stirring. After stirring at 23° C. for 24 h, EtOAc (3 mL) andH₂O (3 mL) were added carefully to the mixture, and the resultingsuspension was evaporated to dryness. After dissolving in H₂O (100 mL),the reaction mixture was washed with Et₂O (2×100 mL). The aqueous layerwas acidified to pH=3 with 10% citric acid solution, and then extractedwith EtOAc (2×100 mL). The combined EtOAc extracts were washedsuccessively with half-saturated NaHCO₃ (150 mL), 5% Na₂S₂O₃ (150 mL),and H₂O (150 mL), dried over Na₂SO₄, and concentrated to giveBoc-NCH₃-(4-F-Phe)-OH as a pale yellow foam (4.37 g, 80%), which wasused without further purification: ¹H NMR (CDCl₃, mixture of isomers) δ1.35 (s), 1.40 (s), 2.69 (s), 2.75 (s), 2.96-3.13 (m), 3.23-3.33 (m),4.54-4.58 (m), 4.76-4.81 (m), 6.96-7.01 (m), 7.15-7.17 (m).

A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) was added to a solutionof ethyl-3-(Boc-L-(Tr-Gln))-E-propenoate (prepared as described inExample 2 above, 3.57 g, 6.73 mmol, 1 equiv.) in the same solvent (15mL) at 23° C. After 2 h, the volatiles were removed under reducedpressure. The residue was dissolved in CH₂Cl₂ (50 mL), andBoc-NCH₃-(4-F-Phe)-OH (prepared as in the preceding paragraph, 2.0 g,6.73 mmol, 1.0 equiv.), HOBt (1.23 g, 9.09 mmol, 1.5 equiv),4-methylmorpholine (2.0 mL, 18.19 mmol, 3 equiv.), and EDC (1.74 g, 9.09mmol, 1.5 equiv.) were added sequentially. The reaction mixture wasstirred at 23° C. overnight, and then was partitioned between water (100mL) and CH₂Cl₂ (2×100 mL). The combined organic layers were dried overNa₂SO₄, concentrated, and the residue was purified by flash columnchromatography (30% EtOAc in hexane) to affordethyl-3-{Boc-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (4.07 g, 84%) aswhite foam: IR (cm⁻¹) 1666, 1510, 1167; ¹H NMR (CDCl₃, mixture ofisomers) δ 1.29 (t, J=7.2), 1.37 (s), 1.65-1.75 (m), 1.95-2.06 (m),2.29-2.33 (m), 2.66 (s), 2.91-2.99 (m), 3.22-3.29 (m), 4.18 (q, J=7.2),4.52-4.58 (m), 5.68 (d, J=15.9), 6.45 (d, J=8.4), 6.74 (dd, J=15.6,5.4), 6.91-6.99 (m), 7.11-7.33 (m); Anal. (C₄₃H₄₈FN₃O₆) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 3 mL), was added to a solutionof ethyl-3-{Boc-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (0.388 g, 0.54mmol, 1 equiv.) in the same solvent (3 mL) at 23° C. After 2 h, thevolatiles were removed under reduced pressure. The residue was dissolvedin DMF (10 mL), cooled at 0° C., and DIEA (0.188 mL, 1.08 mmol, 2equiv.), Boc-L-(2-Naphth)-OH (0.170 g, 0.54 mmol, 1.0 equiv.) and HATU(0.205 g, 0.54 mmol, 1 equiv.) were added sequentially. The reactionmixture was stirred at 23° C. for 1 h. The volatiles were removed underreduced pressure, and the resulting residue was taken into EtOAc (50mL), and washed with 0.5 N HCl (50 mL), saturated NaHCO₃ (50 mL) andbrine (50 mL). The organic layer was dried over Na₂SO₄, concentrated,and the residue was purified by flash column chromatography (40% EtOAcin hexane) to affordethyl-3-{Boc-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate(0.437 g, 88%) as white foam: IR (cm⁻¹) 1656, 1509, 1162; ¹H NMR (CDCl₃,mixture of isomers) δ 0.88 (t, J=7.2), 1.27 (s), 1.30 (s), 1.48-1.58(m), 1.64-1.67 (m), 1.97-2.11 (m), 2.23-2.28 (m), 2.42-2.50 (m),2.62-2.69 (m), 2.80 (s), 2.90 (s), 3.00-3.07 (m), 3.15-3.20 (m),3.25-3.32 (m), 4.18 (q, J=7.2), 4.42-4.46 (m), 4.53-4.56 (m), 4.61-4.66(m), 4.72-4.82 (m), 5.94-5.00 (m), 5.63 (d, J=15.6), 6.12 (d, J=15.6),6.60 (dd, J=15.6, 5.4), 6.75-6.89 (m), 6.70-7.08 (m), 7.19-7.30 (m),7.41-7.50 (m), 7.70-7.82 (m), 8.80 (d, J=8.4); Anal. (C₅₆H₅₉FN₄O₇) C, H,N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-Propenoate

Ethyl-3-{Boc-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate wasconverted to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatein a manner analogous to that described in Example 2 above for theconversion of ethyl-3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate toproductethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoateIR (cm⁻¹) 3296, 1654, 1510; ¹H NMR (DMSO-d₆, mixture of isomers) δ1.17-1.24 (m), 1.62-1.78 (m), 2.04-2.15 (m), 2.38 (s), 2.42 (s),2.71-2.79 (m), 2.84 (s), 2.87-2.92 (m), 3.03 (s), 3.15 (d, J=7.5),3.98-4.06 (m), 4.08-4.12 (m), 4.38-4.42 (m), 4.94 (m), 5.03-5.07 (m),5.09-5.18 (m), 5.66-5.82 (m), 6.42 (s), 6.43 (s), 6.66-6.81 (m),6.88-6.94 (m), 7.01-7.06 (m), 7.13-7.17 (m), 7.24-7.34 (m), 7.43-7.46(m), 7.56 (s), 7.76-7.84 (m), 8.08 (d, J=7.8), 8.60 (d, J=8.4), 9.01 (d,J=7.2); Anal. (C₃₇H₄₀FN₅O₇.0.75H₂O) C, H, N.

Example 14 Preparation of Compound A-9:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-His-NCH₃-L-(4-F-Phe)-L-Gln}-E-Propenoate

Ethyl-3-{Boc-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (described inExample 13 above) was converted to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-His-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatein a manner analogous to the preparation of productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatedescribed in Example 13 above (utilizing Boc-L-(Tr-His)-OH in lieu ofBoc-L-(2-Naphth)-OH): IR (cm⁻¹) 3302, 1665, 1202; ¹H NMR (DMSO-d₆,mixture of isomers) δ 1.21 (t, J=7.2), 1.70-1.78 (m), 2.05-2.09 (m),2.41 (s), 2.44 (s), 2.69-3.26 (m), 4.11 (q, J=7.2), 4.38-4.53 (m),5.07-5.19 (m), 6.51-5.84 (m), 6.44 (s), 6.48 (s), 6.63-6.86 (m),6.89-7.01 (m), 7.09-7.19 (m), 7.23-7.42 (m), 8.02 (d, J=8.7), 8.15 (d,J=8.1), 8.59 (d, J=8.7), 8.94 (s), 9.04 (s), 9.14 (d, J=6.9); Anal.(C₃₀H₃₆FN₇O₇.TFA.H₂O) C, H, N.

Example 15 Preparation of Compound A-12:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Leu-NCH₃-L-(4-F-Phe)-L-Gln}-E-Propenoate

Ethyl-3-{Boc-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (described inExample 13 above) was converted to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Leu-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatein a manner analogous to the preparation of productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatedescribed in Example 13 above (utilizing Boc-L-Leu-OH in lieu ofBoc-L-(2-Naphth)-OH): IR (cm⁻¹) 3325, 1663, 1171; ¹H NMR (DMSO-d₆,mixture of isomers) δ 0.64-0.67 (m), 0.86-0.88 (m), 1.17-1.23 (m),1.32-1.40 (m), 1.59-1.75 (m), 1.98-2.07 (m), 2.42 (s), 2.45 (s), 2.08(s), 2.86-2.93 (m), 2.99 (s), 3.12-3.20 (m), 4.05-4.15 (m), 4.41-4.50(m), 4.82-5.07 (m), 5.62 (d, J=15.9), 5.86 (d, J=15.9), 6.51 (s), 6.53(s), 6.68-6.73 (m), 6.93-6.99 (m), 7.09-7.21 (m), 7.26-7.31 (m), 8.03(d, J=7.8), 8.08 (d, J=7.8), 8.41 (d, J=8.1), 8.94 (d, J=7.2); Anal.(C₃₀H₄₀FN₅O₇.1.25CH₂Cl₂) C, H, N.

Example 16 Preparation of Compound A-13:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-(1-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-Propenoate

Ethyl-3-{Boc-NCH₃-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (described inExample 13 above) was converted to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-(1-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatein a manner analogous to the preparation of productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-(2-Naphth)-NCH₃-L-(4-F-Phe)-L-Gln}-E-propenoatedescribed in Example 13 above, but utilizing Boc-L-(1-Naphth)-OH in lieuof Boc-L-(2-Naphth)-OH: IR (cm⁻¹) 3308, 1659, 1169; ¹H NMR (DMSO-d₆,mixture of isomers) δ 1.16-1.23 (m), 1.61-1.78 (m), 1.98-2.02 (m),2.07-2.12 (m), 2.41 (s), 2.43 (s), 2.77 (s), 2.78 (s), 2.84-2.87 (m),2.93-3.03 (m), 3.08-3.14 (m), 3.31-3.38 (m), 4.00-4.15 (m), 4.27-4.32(m), 4.40-4.46 (m), 4.58-4.64 (m), 5.07-5.17 (m), 5.57-5.73 (m), 6.45(s), 6.57-6.61 (m), 6.71-6.88 (m), 6.89-6.91 (m), 7.11-7.19 (m),7.31-7.38 (m), 7.50-7.58 (m), 7.73-7.78 (m), 7.83-7.94 (m), 8.08 (d,J=8.1), 8.13 (d, J=8.7), 8.62 (d, J=8.1), 9.14 (d, J=8.1); Anal.(C₃₇H₄₀FN₅O₇) C, H, N.

Example 17 Preparation of Compound B-2:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

Preparation of IntermediateEthyl-3-{Boc-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-{(N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala}-E-Propenoate

This intermediate was prepared from{2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamicacid tert-butyl ester (described in Example 10) andethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate(described in Example 6) in a manner analogous to the preparation ofethyl-3-{Boc-L-(4-Me-Phe)-L-(Tr-Gln)}-E-propenoate (Example 4) above:R_(f)=0.24 (60% EtOAc in hexanes); IR (cm⁻¹) 3293, 1717, 1668; ¹H NMR(CDCl₃) δ 0.82 (d, 3H, J=6.8), 1.01 (d, 3H, J=6.8), 1.30 (t, 3H, J=7.2),1.51-1.65 (m, 2H), 1.84-1.96 (m, 1H), 2.16-2.37 (m, 2H), 2.47 (s, 3H),2.49-2.55 (m, 3H), 2.85-3.01 (m, 2H), 3.12-3.25 (m, 3H), 3.77 (s, 3H),3.78 (s, 3H), 4.18 (q, 2H, J=7.2), 4.31-4.49 (m, 3H), 4.65-4.70 (m, 1H),5.53 (dd, 1H, J=15.7, 1.4), 6.39-6.44 (m, 3H), 6.63 (dd, 1H, J=15.7,5.4), 6.93-7.01 (m, 2H), 7.05-7.10 (m, 1H), 7.12-7.18 (m, 2H), 7.24 (d,1H, J=8.7), 7.47 (d, 1H, J=6.5); Anal. (C₄₀H₄₉FN₄O₉.0.5H₂O) C, H, N.

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

Ethyl-3-{Boc-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoatewas converted to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-propenoatein a manner analogous to the conversion ofethyl-3-{Boc-L-Val-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoateto productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-propenoatedescribed in Example 6 above: mp=178-181° C.; R_(f)=0.49 (10% CH₃OH inCHCl₃); IR (cm⁻¹) 3295, 1678 br; ¹H NMR (CDCl₃) δ 0.85 (d, 3H, J=6.8),1.03 (d, 3H, J=6.5), 1.30 (t, 3H, J=7.2), 1.51-1.62 (m, 1H), 1.71-1.93(m, 2H), 2.27-2.40 (m, 2H), 2.47 (s, 3H), 2.51-2.75 (m, 3H), 2.82-2.98(m, 2H), 3.11-3.24 (m, 1H), 3.26-3.42 (m, 2H), 4.18 (q, 2H, J=7.2),4.41-4.53 (m, 1H), 4.63-4.72 (m, 1H), 5.50 (d, 1H, J=15.4), 5.88 (s,1H), 6.39 (s, 1H), 6.63 (dd, 1H, J=15.4, 5.3), 6.92-7.03 (m, 2H),7.08-7.31 (m, 4H); Anal. (C₃₁H₃₉FN₄O₇) C, H, N.

Example 18 Preparation of Compound C-2:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

Preparation of Intermediate(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂

Boc-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂ (prepared as described in Example 12above, 0.91 g, 2.15 mmol) was dissolved in 1,4-dioxane (28 mL), and asolution of HCl in 1,4-dioxane (4.0 M, 14 mL) was added. The reactionwas stirred at room temperature for 14 h. The solvent was removed byevaporation, and the residue taken up in EtOAc (50 mL). The organicphase was washed with saturated NaHCO₃ solution (50 mL) and then brine(50 mL), dried (MgSO₄), and the solvent removed to give 0.66 g (quant.)of a clear oil.

This material was dissolved in CH₂Cl₂ (20 mL). Pyridine (0.17 mL, 2.08mmol) was added, and the reaction was cooled to 0° C.5-Methylisoxazole-3-carbonyl chloride (0.33 g, 2.27 mmol) dissolved in 2mL of CH₂Cl₂ was added, and the reaction warmed to room temperature over1 h. The solvent was removed in vacuo, and the residue purified by flashcolumn chromatography eluting with a gradient of 5→10% EtOAc/hexanes.The (5′-methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂product was obtained as 0.70 g (82%) of a white crystalline solid:R_(f)=0.20 (30% EtOAc/hexanes); IR (KBr) 1745, 1661, 1553, 1186 cm⁻¹; ¹HNMR (DMSO-d₆) δ 0.85-0.92 (m, 6H), 2.14-2.21 (m, 1H), 2.46 (s, 3H),3.05-3.19 (m, 2H), 4.32-4.37 (m, 1H), 4.53-4.60 (m, 2H), 5.16-5.28 (m,3H), 5.74-5.85 (m, 1H), 6.56 (s, 1H), 7.01-7.07 (m, 2H), 7.26-7.29 (m,2H), 8.76 (d, 1H, J=8.1); Anal. (C₂₂H₂₅N₂O₆F) C, H, N.

Preparation of IntermediateEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-Propenoate

(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-OCH₂CH═CH₂ (0.67 g,1.55 mmol) was dissolved in THF (15 mL).Tetrakis(triphenylphosphine)palladium(0) (0.036 g, 0.03 mmol) was added,and the reaction mixture was stirred for 5 minutes. Morpholine (0.16 mL,1.86 mmol) was added dropwise, and the reaction stirred at roomtemperature for 6 h. The solvent was removed in vacuo, and the residuetaken up in 50 mL of Et₂O. The product was extracted twice intosaturated NaHCO₃ solution (50 mL), and the organic phase discarded. Theaqueous phase was acidified to pH=1-2 with 10% HCl, and the productextracted twice with EtOAc (40 mL). The organic phase was washed withbrine (50 mL), dried (MgSO₄), and concentrated to give 0.57 g (95%) ofan oil which crystallized upon standing.

A portion of this material (0.19 g, 0.50 mmol) was dissolved in DMF (3mL). Diisopropylethylamine (0.34 mL, 1.0 mmol) was added, followed byethyl-3-{H₂N-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate.HCl(prepared as described in Examples 4 and 6 above, 0.19 g, 0.50 mmol).The reaction was cooled to 0° C. HATU (0.19 g, 0.50 mmol) was added, andthe reaction was allowed to warm to room temperature. The DMF wasremoved in vacuo. The residue was dissolved with EtOAc(30 mL), and theorganic phase washed consecutively with 10% HCl solution (25 mL),saturated NaHCO₃ solution (25 mL), H₂O (25 mL), and brine (25 mL). Thesolvent was dried (MgSO₄) and filtered, and the residue purified byflash column chromatography (0→1.0% MeOH/CH₂Cl₂) to giveethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(0.27 g, 72%) as a white amorphous solid: R_(f)=0.18 (3% MeOH/CHCl₃);IR(KBr) 1671, 1547, 1510, 1209 cm⁻¹; ¹H NMR (DMSO-d₆) δ 0.88 (d, 3H,J=7.0), 0.93 (d, 3H, J=7.0), 1.20 (t, 3H, J=7.0), 1.41-1.58 (m, 2H),1.79-1.98 (m, 2H), 2.07-2.24 (m, 2H), 2.44 (s, 3H), 3.01-3.13 (m, 4H),3.73 (s, 3H), 3.76 (s, 3H), 4.09 (q, 2H, J=7.0), 4.24 (s, 2H), 4.30 (t,1H, J=7.4), 4.45-4.48 (m, 1H), 5.12 (t, 1H, J=6.3), 5.59 (d, 1H,J=15.8), 6.45 (dd, 1H, J=8.5, 2.2), 6.54-6.56 (m, 2H), 6.73 (dd, 1H,J=15.5, 4.8), 6.89-6.95 (m, 3H), 7.20 (d, 1H, J=8.5), 7.22 (d, 1H,J=8.5), 8.08 (d, 1H, J=8.8), 8.89 (d, 1H, J=7.4).

Preparation of ProductEthyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-Propenoate

Ethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(0.25 g, 0.33 mmol) was dissolved in CHCl₃ (6 mL). Two drops of waterwere added, followed by DDQ (0.10 g, 0.43 mmol). The reaction was heatedto 50-55° C. for 8 h. Upon cooling, the reaction mixture was poured intoEtOAc (30 mL). The organic phase was washed with 30 mL of 2:1 NaHCO₃/1NNaOH solution and then brine (30 mL), dried (MgSO₄), and concentrated.The residue was subjected to flash column chromatography eluting with0→2% MeOH/CH₂Cl₂. Theethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-propenoateproduct was obtained as 0.18 g (90%) of a white amorphous solid:R_(f)=0.09 (3% MeOH/CHCl₃); IR(KBr) 1680, 1549 cm⁻¹; ¹H NMR (DMSO-d₆) δ0.91 (d, 3H, J=6.6), 0.95 (d, 3H, J=6.6), 1.20 (t, 3H, J=7.0), 1.36-1.44(m, 1H), 1.56-1.61 (m, 1H), 1.74-1.82 (m, 1H), 1.94-1.99 (m, 2H),2.18-2.25 (m, 1H), 2.45 (s, 3H), 2.98-3.18 (m, 4H), 4.09 (q, 2H, J=7.0),4.28-4.32 (m, 1H), 4.43-4.46 (m, 1H), 5.12 (m, 1H), 5.58 (d, 1H,J=15.8), 6.56 (s, 1H), 6.72 (dd, 1H, J=15.8, 4.8), 6.91-6.97 (m, 2H),7.21 (d, 1H, J=5.9), 7.23 (d, 1H, J=5.9), 7.59 (s, 1H), 8.06 (d, 1H,J=8.8), 8.92 (d, 1H, J=7.4); Anal. (C₃₀H₃₇N₄O₈F) C, H, N.

Example 19 Preparation of Compound B-3:2-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-(α-Vinyl-γ-Butyrolactone)

Preparation of IntermediateBoc-L-{(N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala}-E-(α-Vinyl-γ-Butyrolactone)

(1S,3′S)-{2-(1′-(2″,4″-Dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylethyl}-carbamicacid tert-butyl ester (0.360 g, 0.881 mmol, 1 equiv.) was oxidized tothe corresponding aldehyde in the manner described in the preparation ofethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(Example 6). This aldehyde was combined with3-(triphenyl-λ⁵-phosphanylidene)-dihydrofuran-2-one (prepared in amanner analogous to that described in Baldwin et al., J. Org. Chem.1971, vol. 36, 1441) (0.320 g, 0.924 mmol, 1.05 equiv.) in a mixture ofethylene glycol dimethyl ether (10 mL) and DMF (2 mL). The reactionmixture was warmed in a 100° C. oil bath for 1.5 h, allowed to cool to23° C. overnight, and then diluted with MTBE (200 mL), washed with water(20 mL) and brine (20 mL), dried over Na₂SO₄ and evaporated. The residuewas purified by flash column chromatography (2.5% CH₃OH in CH₂Cl₂, then67% EtOAc in CH₂Cl₂) to giveBoc-L-{(N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala}-E-(α-vinyl-γ-butyrolactone)as an oil (0.250 g, 60%): R_(f)=0.50 (67% EtOAc in CH₂Cl₂); IR (cm⁻¹)3307, 1754, 1678; ¹H NMR (CDCl₃) δ 1.42 (s, 9H), 1.46-1.68 (m, 2H),2.02-2.13 (m, 1H), 2.18-2.30 (m, 1H), 2.44-2.56 (m, 1H), 2.91-3.04 (m,1H), 3.15-3.27 (m, 3H), 3.80 (s, 6H), 4.34-4.43 (m, 5H), 5.63-5.69 (m,1H), 6.42-6.47 (m, 2H), 6.48-6.53 (m, 1H), 7.09-7.13 (m, 1H).

Preparation of Product2-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-(α-Vinyl-γ-Butyrolactone)

Boc-L-{(N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala}-E-(α-vinyl-γ-butyrolactone)was converted to product2-{(5′-methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-(α-vinyl-γ-butyrolactone)in a manner analogous to that described above for the preparation ofproductethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Pyrrol-Ala)}-E-propenoate(Example 17): R_(f)=0.28 (5% CH₃OH in CH₂Cl₂); IR (cm⁻¹) 3378 br, 1749,1678 br; ¹H NMR (CDCl₃) δ 0.84 (d, 3H, J=6.8), 1.03 (d, 3H, J=6.8),1.45-1.55 (m, 1H), 1.75-2.00 (m, 2H), 2.25-2.43 (m, 2H), 2.46-2.59 (m,2H), 2.48 (s, 3H), 2.63-2.72 (m, 1H), 2.77-2.90 (m, 3H), 3.06-3.26 (m,2H), 3.29-3.44 (m, 2H), 4.32-4.46 (m, 3H), 4.65-4.71 (m, 1H), 5.72 (s,1H), 6.14-6.20 (m, 1H), 6.40 (s, 1H), 6.94-7.02 (m, 2H), 7.03-7.11 (m,2H), 7.24 (d, 1H, J=9.0), 7.60 (d, 1H, J=6.2); Anal. (C₃₁H₃₇FN₄O₇) C, H,N.

Example 20 Preparation of Compound B-4:Ethyl-3-{(5′-Methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-((S)-Piper-Ala)}-E-Propenoate

This product was prepared in analogy to productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-Piper-Ala)}-E-propenoate(Example 9) and productethyl-3-{(5′-methylisoxazole-3′-carbonyl)-L-ValΨ(COCH₂)-L-(4-F-Phe)-L-Gln}-E-propenoate(Example 10) described above: mp=161-162° C.; R_(f)=0.30 (5% CH₃OH inCH₂Cl₂); IR (cm⁻¹) 3295, 1713, 1649; ¹H NMR (CDCl₃) δ 0.84 (d, 3H,J=6.8), 1.03 (d, 3H, J=6.8), 1.30 (t, 3H, J=7.2), 1.43-1.55 (m, 2H),1.77-1.90 (m, 2H), 1.95-2.12 (m, 2H), 2.27-2.38 (m, 1H), 2.44-2.58 (m,2H), 2.48 (s, 3H), 2.66-2.76 (m, 1H), 2.80-2.93 (m, 2H), 3.12-3.42 (m,3H), 4.18 (q, 2H, J=7.2), 4.39-4.49 (m, 1H), 4.65-4.72 (m, 1H), 5.50(dd, 1H, J=15.9, 1.6), 5.80 (s, 1H), 6.38-6.41 (m, 1H), 6.62 (dd, 1H,J=15.9, 5.3), 6.94-7.02 (m, 2H), 7.08-7.28 (m, 4H).

Examples 21 through 30

For Examples 21-30, the following Compounds (A-14) through (A-23),respectively, were prepared using synthetic methods analogous to thosedescribed above for compounds of the formula I-A:

Example 31 Preparation of Comparison Compound #2:Ethyl-3-(Cbz-L-Leu-L-Phe-L-Gln)-E-Propenoate

Preparation of Intermediate [Boc-L-(Tr-Gln)]—N(OMe)Me

Isobutyl chloroformate (4.77 mL, 36.8 mmol, 1.0 equiv.) was added to asolution of N-α-Boc-γ-trityl-L-glutamine (18.7 g, 36.7 mmol, 1 equiv.)and 4-methylmorpholine (8.08 mL, 73.5 mmol, 2.0 equiv.) in CH₂Cl₂ (250mL) at 0° C. The reaction mixture was stirred at 0° C. for 20 minutes,and then N,O-dimethylhydroxylamine hydrochloride (3.60 g, 36.7 mmol, 1.0equiv.) was added. The resulting solution was stirred at 0° C. for 20minutes and at 23° C. for 2 hours, and then was partitioned betweenwater (150 mL) and CH₂Cl₂ (2×150 mL). The combined organic layers weredried over Na₂SO₄ and were concentrated. Purification of the residue byflash column chromatography (gradient elution 40→20% hexanes in EtOAc)provided {Boc-L-(Tr-Gln)}-N(OMe)Me (16.1 g, 82%) as a white foam: IR(cm⁻) 3411, 3329, 3062, 1701, 1659; ¹H NMR (CDCl₃) δ 1.42 (s, 9H),1.63-1.77 (m, 1H), 2.06-2.17 (m, 1H), 2.29-2.43 (m, 2H), 3.17 (s, 3H),3.64 (s, 3H), 4.73 (s, br, 1H), 5.38-5.41 (m, 1H), 7.20-7.31 (m, 15H);Anal. (C₃₁H₃₇N₃O₅) C, H, N.

Preparation of Intermediate {BOC-L-(Tr-Gln)}-H

Diisobutylaluminum hydride (50.5 mL of a 1.5 M solution in toluene, 75.8mmol, 2.5 equiv.) was added to a solution of {Boc-L-(Tr-Gln)}-N(OMe)Me(16.1 g, 30.3 mmol, 1 equiv.) in THF at −78° C., and the reactionmixture was stirred at −78° C. for 4 h. Methanol (4 mL) and 1.0 M HCl(10 mL) were added sequentially, and the mixture was warmed to 23° C.The resulting suspension was diluted with Et₂O (150 mL) and was washedwith 1.0 M HCl (3×100 mL), half-saturated NaHCO₃ (100 mL), and water(100 mL). The organic layer was dried over MgSO₄, filtered, and wasconcentrated to give crude {Boc-L-(Tr-Gln))-H (13.8 g, 97%) as a whitesolid: mp=114-116° C.; IR (cm⁻¹) 3313, 1697, 1494; ¹H NMR (CDCl₃) δ 1.44(s, 9H), 1.65-1.75 (m, 1H), 2.17-2.23 (m, 1H), 2.31-2.54 (m, 2H), 4.11(s, br, 1H), 5.38-5.40 (m, 1H), 7.11 (s, 1H), 7.16-7.36 (m, 15H), 9.45(s, 1H).

Preparation of Intermediate Ethyl-3-{Boc-L-(Tr-Gln)}-E-Propenoate

Sodium bis(trimethylsilyl)amide (22.9 mL of a 1.0 M solution in THF,22.9 mmol, 1.0 equiv.) was added to a solution of triethylphosphonoacetate (5.59 g, 22.9 mmol, 1.0 equiv.) in THF (200 mL) at −78°C., and the resulting solution was stirred for 20 minutes at thattemperature. Crude {Boc-L-(Tr-Gln)}-H (10.8 g, 22.9 mmol, 1 equiv.) inTHF (50 mL) was added via cannula, and the reaction mixture was stirredfor 2 hours at −78° C., warmed to 0° C. for 10 minutes, and partitionedbetween 0.5 M HCl (150 mL) and a 1:1 mixture of EtOAc and hexanes (2×150mL). The combined organic layers were dried over Na₂SO₄ and wereconcentrated. Purification of the residue by flash column chromatography(40% EtOAc in hexanes) provided ethyl-3-{Boc-L-(Tr-Gln)}-E-propenoate(10.9 g, 88%) as a white foam: IR (cm⁻¹) 3321, 1710; ¹H NMR (CDCl₃) δ1.27 (t, 3H, J=7.2), 1.42 (s, 9H), 1.70-1.78 (m, 1H), 1.80-1.96 (m, 1H),2.35 (t, 2H, J=7.0), 4.18 (q, 2H, J=7.2), 4.29 (s, br, 1H), 4.82-4.84(m, 1H), 5.88 (dd, 1H, J=15.7, 1.6), 6.79 (dd, 1H, J=15.7, 5.3), 6.92(s, 1H), 7.19-7.34 (m, 15H); Anal. (C₃₃H₃₈N₂O₅) C, H, N.

Preparation of IntermediateEthyl-3-{Cbz-L-Leu-L-Phe-L-(Tr-Gln)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 20 mL), was added to a solutionof ethyl-3-{Boc-L-(Tr-Gln)}-E-propenoate (1.00 g, 1.84 mmol, 1 equiv.)in the same solvent (20 mL) at 23° C. After 3 hours, the volatiles wereremoved under reduced pressure. The residue was dissolved in CH₂Cl₂ (50mL) and Cbz-L-Leu-L-Phe-OH (0.759 g, 1.84 mmol, 1.0 equiv.), HOBt (0.373g, 2.76 mmol, 1.5 equiv.), 4-methylmorpholine (0.809 mL, 7.36 mmol, 4.0equiv.), and EDC (0.529 g, 2.76 mmol, 1.5 equiv.) were addedsequentially. The reaction mixture was stirred at 23° C. for 18 hours,and then was partitioned between water (150 mL) and EtOAc (2×150 mL).The combined organic layers were dried over Na₂SO₄ and wereconcentrated. Flash chromatographic purification of the residue (5%CH₃OH in CH₂Cl₂) affordedethyl-3-{Cbz-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate (1.25 g, 83%) as awhite solid: mp=192-194° C.; IR (cm⁻¹) 3295, 1696, 1678, 1655, 1519; ¹HNMR (CDCl₃) δ 0.84 (d, 3H, J=6.5), 0.86 (d, 3H, J=6.5), 1.24-1.32 (m,1H), 1.28 (t, 3H, J=7.2), 1.43-1.75 (m, 3H), 1.91-2.06 (m, 1H),2.20-2.38 (m, 2H), 2.93-3.02 (m, 1H), 3.07-3.18 (m, 1H), 3.95-4.02 (m,1H), 4.17 (q, 2H, J=7.2), 4.43-4.55 (m, 2H), 4.82-4.95 (m, 2H), 5.69 (d,1H, J=15.7), 6.46 (d, 1H, J=7.5), 6.60 (d, 1H, J=8.1), 6.69 (dd, 1H,J=15.7, 5.1), 7.09-7.38 (m, 27H); Anal. (C₅₁H₅₆N₄O₇) C, H, N.

Preparation of Product Ethyl-3-(Cbz-L-Leu-L-Phe-L-Gln)-E-Propenoate

Trifluoroacetic acid (20 mL) was added to a solution ofethyl-3-{Cbz-LLeu-L-Phe-L-(Tr-Gln)}-E-propenoate (1.25 g, 1.49 mmol, 1equiv.) and triisopropylsilane (1.53 mL, 7.47 mmol, 5.0 equiv.) inCH₂Cl₂ (20 mL) at 23° C., producing a bright yellow solution. Thereaction mixture was stirred for 20 minutes at 23° C., during which timeit became colorless. Carbon tetrachloride (20 mL) was added, and thevolatiles were removed under reduced pressure. The residue wastriturated with Et₂O (20 mL), and the resulting white solid wascollected by vacuum filtration, washed with Et₂O (3×50 mL), andair-dried to afford ethyl-3-(Cbz-L-Leu-L-Phe-L-Gln)-E-propenoate (0.717g, 81%): mp=219-221° C.; IR (cm⁻¹) 3300, 1672, 1535; ¹H NMR (DMSO-d₆) δ0.78 (d, 3H, J=6.8), 0.82 (d, 3H, J=6.5), 1.21 (t, 3H, J=7.0), 1.25-1.37(m, 2H), 1.42-1.54 (m, 1H), 1.58-1.80 (m, 2H), 2.02-2.09 (m, 2H), 2.84(dd, 1H, J=13.2, 8.9), 2.97 (dd, 1H, J=13.2, 5.8), 3.93-4.01 (m, 1H),4.11 (q, 2H, J=7.0), 4.33-4.52 (m, 2H), 4.97 (d, 1H, J=12.3), 5.04 (d,1H, J=12.3), 5.64 (d, 1H, J=15.9), 6.69 (dd, 1H, J=15.9, 5.4), 6.76 (s,1H), 7.13-7.37 (m, 11H), 7.43 (d, 1H, J=7.8), 7.99 (d, 1H, J=8.1), 8.04(d, 1H, J=8.1); Anal. (C₃₂H₄₂N₄O₇) C, H, N.

Example 32 Preparation of Comparison Compound #3:Ethyl-3-(Cbz-L-Val-L-Phe-L-Gln)-E-Propenoate

Preparation of Intermediate Ethyl-3-{Boc-L-Phe-L-(Tr-Gln)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 10 mL) was added to a solutionof ethyl-3-{Boc-L-(Tr-Gln)}-E-propenoate (prepared as described inExample 31, 3.05 g, 5.62 mmol, 1 equiv.) in the same solvent (20 mL) at23° C. After 3 hours, the volatiles were removed under reduced pressure.The residue was dissolved in CH₂Cl₂ (50 mL), and Boc-L-Phe-OH (1.49 g,5.62 mmol, 1.0 equiv.), HOBt (0.911 g, 6.74 mmol, 1.2 equiv.),4-methylmorpholine (1.85 mL, 16.8 mmol, 3.0 equiv.) and EDC (1.29 g,6.73 mmol, 1.2 equiv.) were added sequentially. The reaction mixture wasstirred at 23° C. for 18 hours, and was then partitioned between water(150 mL) and EtOAc (2×150 mL). The combined organic layers were driedover Na₂SO₄ and were concentrated. Flash chromatographic purification ofthe residue (gradient elution, 40→50% EtOAc in hexanes) affordedethyl-3-{Boc-L-Phe-L-(Tr-Gln)}-E-propenoate (2.77 g, 71%) as a whitefoam: IR (cm⁻¹) 3306, 1706, 1661; ¹H NMR (CDCl₃) δ 1.29 (t, 3H, J=7.2),1.38 (s, 9H), 1.65-1.76 (m, 1H), 1.87-1.99 (m, 1H), 2.25-2.27 (m, 2H),2.94-3.01 (m, 2H), 4.14-4.26 (m, 3H), 4.48-4.53 (m, 1H), 4.95 (s, br,1H), 5.64 (d, 1H, J=15.8), 6.29 (d, 1H, J=8.1), 6.64 (dd, 1H, J=15.8,5.4), 6.80 (s, br, 1H), 7.14-7.32 (m, 20H); Anal. (C₄₂H₄₇N₃O₆) C, H, N.

Preparation of IntermediateEthyl-3-{Cbz-L-Val-L-Phe-L-(Tr-Gln)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 8 mL) was added to a solutionof ethyl-3-{Boc-L-Phe-L-(Tr-Gln)}-E-propenoate (0.296 g, 0.429 mmol, 1equiv.) in the same solvent (10 mL) at 23° C. After 3 hours, thevolatiles were removed under reduced pressure. The residue was dissolvedin CH₂Cl₂ (10 mL), and then Cbz-L-Val-OH (0.108 g, 0.430 mmol, 1.0equiv.), HOBt (0.070 g, 0.518 mmol, 1.2 equiv.), 4-methylmorpholine(0.142 mL, 1.29 mmol, 3.0 equiv.) and EDC (0.099 g, 0.516 mmol, 1.2equiv.) were added sequentially. The reaction mixture was stirred at 23°C. for 4 hours, and then was partitioned between water (100 mL) andEtOAc (2×100 mL). The combined organic layers were dried over Na₂SO₄ andwere concentrated. Flash chromatographic purification of the residue (3%CH₃OH in CH₂Cl₂) affordedethyl-3-{Cbz-L-Val-L-Phe-L-(Tr-Gln)}-E-propenoate (0.220 g, 62%) as awhite solid: mp=195-198° C.; IR (cm⁻¹) 3284, 1689, 1646; ¹H NMR (CDCl₃)δ 0.69 (d, 3H, J=6.9), 0.82 (d, 3H, J=6.5), 1.28 (t, 3H, J=7.2),1.63-1.74 (m, 1H), 1.96-2.02 (m, 2H), 2.22-2.35 (m, 2H), 2.93 (dd, 1H,J=14.0, 7.6), 3.10 (dd, 1H, J=14.0, 6.7), 3.81-3.85 (m, 1H), 4.17 (q,2H, J=7.2), 4.48-4.58 (m, 2H), 4.87 (d, 1H, J=12.0), 4.94 (d, 1H,J=12.0), 5.06 (d, 1H, J=6.9), 5.67 (d, 1H, J=15.6), 6.43 (d, 1H, J=7.5),6.63-6.72 (m, 2H), 7.10-7.40 (m, 26H); Anal. (C₅₀H₅₄N₄O₇) C, H, N.

Preparation of Product Ethyl-3-(Cbz-L-Val-L-Phe-L-Gln)-E-Propenoate

Trifluoroacetic acid (5 mL) was added to a solution ofethyl-3-{Cbz-L-Val-L-Phe-L-(Tr-Gln)}-E-propenoate (0.188 g, 0.229 mmol,1 equiv.) and triisopropylsilane (0.300 mL, 1.46 mmol, 6.4 equiv.) inCH₂Cl₂ (10 mL) at 23° C., producing a bright yellow solution. Thereaction mixture was stirred for 20 min. at 23° C., during which time itbecame colorless. Carbon tetrachloride (10 mL) was added, and thevolatiles were removed under reduced pressure. The residue wastriturated with Et₂O (20 mL), and the resulting white solid wascollected by vacuum filtration, washed with Et₂O (3×50 mL), andair-dried to afford ethyl-3-(Cbz-L-Val-L-Phe-L-Gln)-E-propenoate (0.094g, 71%): mp=240° C. (dec); IR (cm⁻¹) 3263, 1686, 1640; ¹H NMR (DMSO-d₆)δ 0.73 (d, 6H, J=6.9), 1.21 (t, 3H, J=7.2), 1.60-1.75 (m, 2H), 1.83-1.90(m, 1H), 2.03-2.08 (m, 2H), 2.83 (dd, 1H, J 13.6, 8.6), 2.96 (dd, 1H,J=13.6, 6.2), 3.79-3.84 (m, 1H), 4.10 (q, 2H, J=7.2), 4.37-4.49 (m, 1H),4.51-4.56 (m, 1H), 4.99 (d, 1H, J=12.5), 5.06 (d, 1H, J=12.5), 5.61 (d,1H, J=15.5), 6.67 (dd, 1H, J=15.5, 5.5), 6.76 (s, 1H), 7.13-7.36 (m,12H, 8.06 (d, 2H, J=8.1); Anal. (C₃₁H₄₀N₄O₇) C, H, N.

Example 33 Preparation of Compound (A-24):Ethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Pyrrol-Ala)}-E-Propenoate

Preparation of IntermediateEthyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-Dimethoxybenzyl)(S)-Pyrrol-Ala)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 4 mL) was added to a solutionof ethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrol-Ala)}-E-propenoate(prepared as described in Example 6) (0.139 g, 0.292 mmol, 1 equiv.) in1,4-dioxane (4 mL). After stirring 1.5 h, the volatiles were evaporatedto give the crude amine salt, which was used without purification.

This amine salt was dissolved in CH₂Cl₂ (7 mL), and thenCbz-L-Leu-L-Phe-OH (0.156 g, 0.378 mmol, 1.3 equiv.), 4-methylmorpholine(0.128 mL, 1.16 mmol, 4 equiv.), HOBt (0.067 g, 0.50 mmol, 1.7 equiv.)and EDC (0.095 g, 0.50 mmol, 1.7 equiv) were added sequentially. Afterstirring 20 hours, the reaction mixture was poured into brine (15 mL)and extracted with 10% CH₃OH in CH₂Cl₂ (3×25 mL). The combined organicphases were dried over Na₂SO₄ and evaporated. Purification of theresidue by flash column chromatography (60% EtOAc in hexanes) providedethyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(0.158 g, 70%) as a foam: ¹H NMR (CDCl₃) δ 0.87-0.92 (m, 6H), 1.28 (t,3H, J=7.2), 1.46-1.68 (m, 5H), 1.74-1.86 (m, 1H), 1.97-2.19 (m, 2H),3.02 (dd, 1H, J=13.7, 5.6), 3.11-3.24 (m, 3H), 3.78 (s, 3H), 3.79 (s,3H), 4.17 (q, 2H, J=7.2), 4.20-4.30 (m, 2H), 4.35-4.45 (m, 2H),4.82-4.90 (m, 1H), 5.07 (d, 1H, J=12.3), 5.13 (d, 1H, J=12.3), 5.36 (d,1H, J=7.8), 5.82 (dd, 1H, J=15.6, 1.2), 6.42-6.46 (m, 2H), 6.72 (dd, 1H,J=15.6, 5.3), 6.88 (d, 1H, J=8.7), 7.09 (d, 1H, J=9.0), 7.13-7.20 (m,5H), 7.29-7.37 (m, 5H), 8.09 (d, 1H, J=6.5).

Preparation of ProductEthyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Pyrrol-Ala)}-E-Propenoate

Ethyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(0.156 g, 0.202 mmol, 1 equiv.) and ammonium cerium(IV) nitrate (0.277g, 0.505 mmol, 2.5 equiv.) were combined in a mixture of THF/water 2:1(3 mL) and stirred 2 hours. The reaction mixture was poured into brine(15 mL) and extracted with 10% CH₃OH in CH₂Cl₂ (3×25 mL). The combinedorganic phases were dried over Na₂SO₄ and evaporated. Purification ofthe residue by flash column chromatography (gradient elution, 2→5% CH₃OHin CH₂Cl₂) providedethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Pyrrol-Ala)}-E-propenoate (0.059 g, 47%)as an off-white solid: ¹H NMR (CDCl₃) δ 0.85-0.92 (m, 6H), 1.28 (t, 3H,J=7.2), 1.39-1.65 (m, 4H), 1.68-1.93 (m, 2H), 2.08-2.20 (m, 1H),2.27-2.38 (m, 1H), 3.02-3.13 (m, 2H), 3.24-3.32 (m, 2H), 4.11-4.20 (m,1H), 4.18 (q, 2H), J=7.2), 4.47-4.58 (m, 1H), 4.81-4.89 (m, 1H), 5.05(d, 1H, J=12.1), 5.12 (d, 1H, J=12.1), 5.26 (d, 1H, J=8.1), 5.78 (dd,1H, J=15.7, 1.2), 6.23 (s, 1H), 6.72 (dd, 1H, J=15.7, 5.3), 7.13-7.25(m, 6H), 7.30-7.37 (m, 5H), 7.54 (d, 1H, J=7.2).

Example 34 Preparation of Compound (A-25):Ethyl-3-{Cbz-L-Val-L-Phe-L-((S)-Pyrrol-Ala)}-E-Propenoate

Preparation of IntermediateEthyl-3-{Cbz-L-Val-L-Phe-L-((N-2,4-Dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-Propenoate

In a manner analogous to that used for the conversion ofethyl-3-{Boc-L-(Tr-Gln)}-E-propenoate toethyl-3-{Cbz-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate described in Example31,ethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(prepared as in Example 33) was coupled with Cbz-L-Val-L-Phe-OH toaffordethyl-3-{Cbz-L-Val-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate:IR (cm¹) 3288, 1699,1652; ¹H NMR (CDCl₃) δ 0.87 (d, 3H, J=6.8), 0.95 (d,3H, J=6.5), 1.28 (t, 3H, J=7.2), 1.48-1.60 (m, 2H), 1.70-1.84 (m, 1H),1.95-2.20 (m, 3H), 3.01 (dd, 1H, J=13.4, 5.6), 3.09-3.25 (m, 3H), 3.78(s, 3H), 3.80 (s, 3H), 4.03-4.10 (m, 1H), 4.17 (q, 2H, J=7.2), 4.24 (d,1H, J=14.2), 4.33-4.44 (m, 1H), 4.38 (d, 1H, J=14.2), 4.85-4.94 (m, 1H),5.08 (d, 1H, J=12.1), 5.14 (d, 1H, J=12.1), 5.39 (d, 1H, J=8.1), 5.80(dd, 1H, J=15.6, 1.2), 6.42-6.47 (m, 2H), 6.70 (dd, 1H, J=15.6, 5.3),6.81 (d, 1H, J=9.0), 7.11-7.20 (m, 6H), 7.31-7.39 (m, 5H), 8.11 (d, 1H,J=6.2); Anal. (C₄₂H₅₂N₄O₉) C, H, N.

Preparation of ProductEthyl-3-{Cbz-L-Val-L-Phe-L-((S)-Pyrrol-Ala)}-E-Propenoate

A suspension ofethyl-3-{Cbz-L-Val-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoate(0.215 g, 0.284 mmol, 1 equiv.), DDQ (0.071 g. 0.31 mmol, 1.1 equiv.)and water (3 drops) in CHCl₃ (4 mL) was stirred 1 h at 23° C., and wasthen warmed to reflux for 6 h. After cooling overnight, more DDQ (0.019g, 0.084 mmol, 0.3 equiv.) was added, and the mixture was warmed to 67°C. for 1 h and then evaporated. Purification of the residue by flashcolumn chromatography (gradient elution, 2→5% CH₃OH in CH₂Cl₂) providedslightly impure material, which was dissolved in CH₂Cl₂ (70 mL) andwashed with saturated NaHCO₃ (2×30 mL) and brine (30 mL), and then driedover Na₂SO₄ and evaporated. The residue was stirred in Et₂O (10 mL) for20 minutes, and the solid was collected by filtration and dried undervacuum to provideethyl-3-{Cbz-L-Val-L-Phe-L-((S)-Pyrrol-Ala)}-E-propenoate (0.060 g, 35%)as a an off-white solid: mp=215-217° C.; IR (cm⁻¹) 3413, 3295, 1696,1649; ¹H NMR (CDCl₃) δ 0.83 (d, 3H, J=6.5), 0.91 (d, 3H, J=6.8), 1.28(t, 3H, J=7.2), 1.50-1.59 (m, 1H), 1.70-1.91 (m, 2H), 2.03-2.17 (m, 2H),2.26-2.38 (m, 1H), 3.03 (dd, 1H, J=13.5, 6.4), 3.12 (dd, 1H, J=13.5,6.4), 3.21-3.34 (m, 2H), 3.96 (dd, 1H, J=8.3, 6.4), 4.17 (q, 2H, J=7.2),4.45-4.56 (m, 1H), 4.83-4.92 (m, 1H), 5.07 (d, 1H, J=12.1), 5.13 (d, 1H,J=12.1), 5.29 (d, 1H, J=8.3), 5.77 (dd, 1H, J=15.8, 1.2), 5.94 (s, 1H),6.71 (dd, 1H, J=15.8, 5.3), 6.95 (d, 1H, J=9.0), 7.14-7.27 (m, 5H),7.31-7.38 (m, 5H), 7.57 (d, 1H, J=7.2); Anal. (C₃₃H₄₂N₄O₇) C, H, N.

Example 35 Preparation of Compound (A-26):Ethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Piper-Ala)}-E-Propenoate

Preparation of ProductEthyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Piper-Ala)}-E-Propenoate

(1S,3′S)-{2-(1′-(2″,4″-Dimethoxybenzyl)-2′-oxo-piperidin-3′-yl)-1-hydroxy-methylethyl}-carbamicacid tert-butyl ester (prepared as described in Example 8) was convertedto the product ethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Piper-Ala)}-E-propenoatein a manner analogous to the conversion of(1S,3′S)-{2-(1′-(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethyl-ethyl}-carbamicacid tert-butyl ester to productethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Pyrrol-Ala)}-E-propenoate as describedin Example 34: IR (cm⁻¹) 3422, 3307, 1713, 1649; ¹H NMR (CDCl₃) δ0.86-0.92 (m, 6H), 1.28 (t, 3H, J=7.2), 1.38-1.75 (m, 6H), 1.77-1.89 (m,1H), 1.96-2.11 (m, 2H), 3.07 (d, 2H, J=6.2), 3.20-3.27 (m, 2H),4.13-4.24 (m, 1H), 4.18 (q, 2H, J=7.2), 4.41-4.53 (m, 1H), 4.76-4.85 (m,1H), 5.06 (d, 1H, J=12.1), 5.12 (d, 1H, J=12.1), 5.34 (d, 1H, J=7.8),5.78 (dd, 1H, J=15.6, 5.4), 6.17 (s, 1H), 6.70 (dd, 1H, J=15.6, 5.4),7.00 (d, 1H, J=8.4), 7.13-7.27 (m, 6H), 7.30-7.37 (m, 5H), 7.83 (d, 1H,J=6.8); Anal. (C₃₅H₄₆N₄O₇.0.5H₂O) C, H, N.

Example 36 Preparation of Compound (A-27):Ethyl-3-{Cbz-L-Leu-L-Phe-L-((R)-Pyrrol-Ala)}-E-Propenoate

Preparation of Intermediate(4S,4″R)-4-{3′-(4″-Benzyl-2″-oxo-oxazolidin-3″-yl)-3′-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylicAcid tert-Butyl Ester

Triethylamine (6.43 mL, 46.1 mmol, 3.0 equiv.) and pivaloyl chloride(1.89 mL, 15.3 mmol, 1.0 equiv.) were added sequentially to a solutionof (4S)-4-(2′-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acidtert-butyl ester (4.20 g, 15.3 mmol, 1 equiv.) in THF (300 mL) at 0° C.The cloudy reaction mixture was stirred at 0° C. for 3.5 h, and thenlithium chloride (0.716 g, 16.9 mmol, 1.1 equiv.) and(R)-(+)-4-benzyl-2-oxazolidinone (2.59 g, 14.6 mmol, 0.95 equiv.) wereadded sequentially. After warming to 23° C. and stirring for 19 h, thereaction mixture was partitioned between 0.5 M HCl (150 mL) and EtOAc(2×150 mL). The combined organic layers were washed with half-saturatedNa₂CO₃ (150 mL), dried over MgSO₄, and gravity filtered. The filtratewas concentrated under reduced pressure and the residue was purified byflash column chromatography (30% EtOAc in hexanes) to give(4S,4″R)-4-{3′-(4″-benzyl-2″-oxo-oxazolidin-3″-yl)-3′-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (6.15 g, 97%) as a colorless oil: IR (cm⁻¹) 2978,1783, 1694; ¹H NMR (CDCl₃, mixture of isomers) δ 1.46 (s), 1.58 (s),1.63 (s), 2.01-2.05 (m), 2.72-3.13 (m), 3.29-3.33 (m), 3.74-3.79 (m),3.82-4.09 (m), 4.11-4.25 (m), 4.67-4.70 (m), 7.20-7.37 (m); Anal.(C₂₃H₃₂N₂O₆) C, H, N.

Preparation of Intermediate(2′R,4S,4″R)-4-{2′-(4″-Benzyl-2″-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl}-2,2-dimethyloxazolidine-3-carboxylicAcid tert-Butyl Ester

A solution of(4S,4″R)-4-{3′-(4″-benzyl-2″-oxo-oxazolidin-3′-yl)-3′-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (6.15 g, 14.2 mmol, 1 equiv.) in THF (25 mL) wasadded to a solution of sodium bis(trimethylsilyl)amide (14.2 mL of a 1.0M solution in THF, 14.2 mmol, 1.0 equiv.) in the same solvent (50 mL) at−78° C. The reaction mixture was stirred for 15 minutes at −78° C., andthen allyl iodide (3.90 mL, 42.6 mmol, 3.0 equiv.) was added. Afterstirring an additional 2 h at −78° C., the reaction mixture wasmaintained at −45° C. for 2 h, and then was partitioned between a 2:1mixture of half-saturated NH₄Cl and 5% Na₂S₂O₃ (200 mL) and a 1:1mixture of EtOAc and hexanes (2×150 mL). The combined organic layerswere washed with H₂O (100 mL), dried over MgSO₄, and gravity filtered.The filtrate was concentrated under reduced pressure and the residue waspurified by flash column chromatography (15% EtOAc in hexanes) toprovide(2′R,4S,4″R)-4-{2′-(4″-benzyl-2″-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (3.12 g, 46%) as a viscous foam: IR (cm⁻¹) 2978,1782, 1685; ¹H NMR (CDCl₃, mixture of isomers) δ 1.42 (s), 1.45 (s),1.49 (s), 1.52 (s), 1.62-1.78 (m), 1.80-2.01 (m), 2.23-2.49 (m),2.51-2.56 (m), 2.76 (dd, J=13.3, 9.7), 3.26 (dd, J=13.3, 3.6), 3.58-3.64(m), 3.67 (d, J=8.7), 3.90-3.98 (m), 4.02-4.15 (m), 4.16-4.30 (m),4.75-4.82 (m), 5.06-5.11 (m), 5.74-5.88 (m), 7.22-7.36 (m); Anal.(C₂₆H₃₆N₂O₆) C, H, N.

Preparation of Intermediate(1S,3′R)-{2-(1′-(2″,4″-Dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylethyl}-carbamicAcid tert-Butyl Ester

Ozone was bubbled through a solution of(2′R,4S,4″R)-4-{2′-(4″-benzyl-2″-oxo-oxazolidine-3″-carbonyl)-pent-4′-enyl}-2,2-dimethyloxazolidine-3-carboxylicacid tert-butyl ester (3.12 g, 6.60 mmol, 1 equiv.) in CH₂Cl₂ (200 mL)and CH₃OH (0.535 mL, 13.2 mmol, 2.0 equiv.) at −78° C. until a bluecolor persisted. The reaction mixture was then purged with argon untilit became colorless. Methyl sulfide (4.85 mL, 66.0 mmol, 10 equiv.) wasadded, and the mixture was stirred at −78° C. for 3.5 h and then wasmaintained at 0° C. for an additional 1 h. After partitioning thereaction mixture between H₂O (150 mL) and a 1:1 mixture of EtOAc andhexanes (2×150 mL), the combined organic layers were dried over MgSO₄and gravity filtered. The filtrate was concentrated under reducedpressure, and the residue was immediately utilized without furtherpurification.

The above residue was dissolved in a 2:1 mixture of THF, and then EtOH(180 mL) at 23° C. and 2,4-dimethoxybenzylamine hydrochloride (5.38 g,26.4 mmol, 4.0 equiv.), sodium acetate (2.17 g, 26.4 mmol, 4.0 equiv.),and sodium cyanoborohydride (0.829 g, 13.2 mmol, 2.0 equiv.) were addedsequentially. The resulting suspension was stirred for 19 h at 23° C.,and then was partitioned between 0.5 M HCl (150 mL) and EtOAc (2×100mL). The combined organic layers were washed with half-saturated NaHCO₃(100 mL), dried over Na₂SO₄, and concentrated under reduced pressure.The residue was passed through a short silica gel column (eluting with50% EtOAc in hexanes) to give(3′R,4S)-4-{1′-(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′ylmethyl}-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester contaminated with(R)-(+)-4-benzyl-2-oxazolidinone.

This material was dissolved in CH₃OH (80 mL), and TsOH.H₂O (0.251 g,1.32 mmol, 0.20 equiv.) was added. The reaction mixture was heated to50° C. and was maintained at that temperature for 4 h. After cooling to23° C., the reaction mixture was concentrated under reduced pressure to˜20 mL volume and was partitioned between half-saturated NaHCO₃ (150 mL)and a 9:1 mixture of CH₂Cl₂ and CH₃OH (2×150 mL). The combined organiclayers were dried over Na₂SO₄ and concentrated under reduced pressure.Purification of the residue by flash column chromatography (3% CH₃OH inCH₂Cl₂) afforded(1S,3′R)-{2-(1′-(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylethyl}-carbamicacid tert-butyl ester (0.92 g, 34%) as a foam: IR (cm⁻¹) 3347 (br),2937, 1669; ¹H NMR (CDCl₃) δ 1.44 (s, 9H), 1.62-1.77 (m, 2H), 1.94-2.04(m, 1H), 2.15-2.26 (m, 1H), 2.40-2.50 (m, 1H), 3.13-3.24 (m, 2H),3.56-3.77 (m, 3H), 3.80 (s, 6H), 3.82-4.16 (m, 1H), 4.37 (d, 1H,J=14.3), 4.45 (d, 1H, J=14.3), 5.49 (d, 1H, J=7.8), 6.42-6.45 (m, 2H),7.08-7.11 (m, 1H); Anal. (C₂₁H₃₂N₂O₆.0.25H₂O) C, H, N.

Preparation of IntermediateEthyl-3-{Boc-L-((N-2,4-Dimethoxybenzyl)-(R)-Pyrrol-Ala)}-E-Propenoate

In a manner analogous to the conversion of(1S,3′S)-{2-(1′-(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl}1-hydroxymethylethyl}carbamicacid tert-butyl ester toethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoatedescribed in Example 33,(1S,3′R){2-(1′(2″,4″-dimethoxybenzyl)-2′-oxo-pyrrolidin-3′-yl)-1-hydroxymethylethyl}-carbamicacid tert-butyl ester was transformed intoethyl-3-{Boc-L-(N-2,4-dimethoxybenzyl)-(R)-Pyrrol-Ala)}-E-propenoate: IR(cm⁻¹) 3307, 1713, 1674; ¹H NMR (CDCl₃) δ 1.28 (t, 3H, J=7.2), 1.45 (s,9H), 1.57-1.82 (m, 2H), 2.02-2.19 (m, 2H), 2.42-2.55 (m, 1H), 3.11-3.25(m, 2H), 3.79 (s, 3H), 3.80 (s, 3H), 4.19 (q, 2H, J=7.2), 4.32-4.50 (m,3H), 5.97 (dd, 1H, J=15.7, 1.4), 6.38 (d, 1H, J=7.8), 6.42-6.47 (m, 2H),6.86 (dd, 1H, J=15.7, 5.1), 7.08-7.13 (m, 1H); Anal. (C₂₅H₃₆N₂O₇) C, H,N.

Preparation of IntermediateEthyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-Dimethoxybenzyl)-(R)-Pyrrol-Ala)}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 6 mL) was added to a solutionof ethyl-3-{Boc-L-((N-2,4-dimethoxybenzyl)-(R)-Pyrrol-Ala)}-E-propenoate(0.233 g, 0.489 mmol, 1 equiv.) in 1,4-dioxane (6 mL). After stirring1.5 h, the volatiles were evaporated to give the crude amine salt, whichwas used without purification.

This amine salt was dissolved in DMF (4 mL) and cooled to 0° C.Cbz-L-Leu-L-Phe-OH (0.202 g, 0.490 mmol, 1 equiv.), DIEA (0.255 mL, 1.46mmol, 3 equiv.) and HATU (0.186 g, 0.489 mmol, 1 equiv.) were addedsequentially. After stirring 1.5 h, the reaction mixture was dilutedwith MTBE (100 mL), washed with 5% KHSO₄, saturated NaHCO₃ and brine (10mL), dried over MgSO₄, and evaporated. Purification of the residue byflash column chromatography (gradient elution, 60→75% EtOAc in hexanes)providedethyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(R)-Pyrrol-Ala)}-E-propenoate(0.208 g, 55%) as a white solid (after evaporation from Et₂O):mp=174-176° C.; IR (cm⁻¹) 3287, 1713, 1649; ¹H NMR (CDCl₃) δ 0.84-0.91(m, 6H), 1.29 (t, 3H, J=7.2), 1.42-1.66 (m, 4H), 1.67-1.77 (m, 1H),1.84-1.95 (m, 1H), 2.20-2.12 (m, 1H), 2.33-2.45 (m, 1H), 3.04-3.23 (m,4H), 3.78 (s, 3H), 3.79 (s, 3H), 4.15-4.29 (m, 1H), 4.17 (q, 2H, J=7.2),4.31 (d, 1H, J=14.5), 4.40 (d, 1H, J=14.5), 4.67-4.84 (m, 2H), 5.05 (d,1H, J=12.1), 5.11 (d, 1H, J=12.1), 5.35 (d, 1H, J=8.1), 5.76 (dd, 1H,J=15.6, 1.6), 6.42-6.46 (m, 2H), 6.74-6.81 (m, 1H), 6.75 (dd, 1H,J=15.6, 5.0), 7.06-7.10 (m, 1H), 7.15-7.24 (m, 5H), 7.29-7.36 (m, 5H),8.79 (d, 1H, J=5.9); Anal. (C₄₃H₅₄N₄O₉) C, H, N.

Preparation of ProductEthyl-3-{Cbz-L-Leu-L-Phe-L-((R)-Pyrrol-Ala)}-E-Propenoate

Ethyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(R)-Pyrrol-Ala)}-E-propenoatewas converted to the productethyl-3-{Cbz-L-Leu-L-Phe-L-((R)-Pyrrol-Ala)}-E-propenoate in a manneranalogous to the conversion ofethyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-Pyrrol-Ala)}-E-propenoateto product ethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-Pyrrol-Ala)}-E-propenoatedescribed in Example 34: IR (cm⁻¹) 3290, 1702, 1642; ¹H NMR (CDCl₃) δ0.85-0.92 (m, 6H), 1.30 (t, 3H, J=7.2), 1.35-1.49 (m, 1H), 1.52-1.71 (m,3H), 1.73-1.94 (m, 2H), 2.15-2.26 (m, 1H), 2.32-2.43 (m, 1H), 3.02-3.18(m, 2H), 3.19-3.29 (m, 2H), 4.15-4.27 (m, 1H), 4.18 (q, 2H, J=7.2),4.65-4.74 (m, 1H), 4.76-4.85 (m, 1H), 5.07 (d, 1H, J=12.3), 5.12 (d, 1H,J=12.3), 5.18-5.25 (m, 1H), 5.76-5.84 (m, 2H), 6.64-6.78 (m, 2H),7.15-7.40 (m, 10H), 7.91-7.98 (m, 1H); Anal. (C₃₄H₄₄N₄O₇) C, H, N.

Example 37 Preparation of Compound (A-28):Ethyl-3-{Cbz-L-Leu-L-Phe-L-1-(2-imidazolidone)Ala}-E-Propenoate

Preparation of IntermediateEthyl-3-{Cbz-L-Leu-L-Phe-L-Boc-aminoAla}-E-Propenoate

(Carbethoxymethlene)triphenylphosphorane (1.20 g, 3.28 mmol, 1.2 equiv.)was added to a solution of Cbz-L-Leu-L-Phe-L-N-Boc-aminoalaninal(prepared as described in Webber et al., J. Med. Chem. 1998, vol. 41,2786) (1.60 g, 2.73 mmol, 1 equiv.) in THF (55 mL), and the reaction wasstirred at room temperature overnight. The volatiles were then removedin vacuo, and the residue was purified by flash column chromatographyeluting (gradient elution, 0→1.5% CH₃OH in CH₂Cl₂) to giveethyl-3-{Cbz-L-Leu-L-Phe-L-N-Boc-aminoAla}-E-propenoate (0.968 g,contaminated with triphenylphosphine oxide). This material was usedwithout further purification.

Preparation of IntermediateEthyl-3-{Cbz-L-Leu-L-Phe-L-(2-Boc-2-aminoethyl)aminoAla}-E-Propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 10 mL) was added to a solutionof ethyl-3-{Cbz-L-Leu-L-Phe-L-Boc-aminoAla}-E-propenoate (0.95 g, 1.46mmol, 1 equiv.) in the same solvent (20 mL) at 23° C. The reactionmixture was stirred at that temperature for 1.5 h, and then additionalHCl in 1,4-dioxane (4.0 M, 10 mL) was added. After stirring overnight,the volatiles were removed in vacuo and the residue was triturated withEt₂O (20 mL). The resulting solids were filtered and were washed withEt₂O (3×10 mL) to give the crude amine salt (0.63 g 73%, 1.05 mmol) as awhite solid.

This material was dissolved in CH₃OH (10 mL), and thenN-Boc-2-aminoethanal (prepared as described in Bischofberger et al., J.Org. Chem. 1988, vol. 53, 3457) (0.19 g, 1.16 mmol, 1.1 equiv.) andNaBH₃CN (0.069 g, 1.05 mmol, 1.0 equiv.) were added sequentially. Thereaction mixture was stirred at 23° C. overnight, and then the volatileswere removed under reduced pressure. The residue was dissolved in EtOAc(25 mL) and the organic layer was washed with water (25 mL) and brine(25 mL), and then dried over MgSO₄ and concentrated. The residue waspurified by flash column chromatography (gradient elution, 0→3% CH₃OH inCH₂Cl₂) to provideethyl-3-{Cbz-L-Leu-L-Phe-L-(2-Boc-2-aminoethyl)aminoAla}-E-propenoate(0.32 g, 44%) as a white amorphous solid: R_(f)=0.20 (5% CH₃OH inCHCl₃); IR (cm⁻¹) 1712, 1649, 1537, 1252, 1175; ¹H NMR (DMSO-d₆) δ 0.79(d, 3H, J=6.6), 0.82 (d, 3H, J=6.6), 1.21 (t, 3H, J=7.0), 1.26-1.37 (m,13H), 1.46-1.54 (m, 1H), 2.56-2.60 (m, 2H), 2.82-2.97 (m, 4H), 3.98-4.04(m, 1H), 4.10 (q, 2H, J=7.0), 4.42-4.49 (m, 2H), 4.98 (d, 1H, J=12.5),5.04 (d, 1H, J=12.9), 5.59 (d, 1H, J=15.8), 6.73-6.75 (m, 1H), 6.77 (dd,1H, J=15.8, 4.8), 7.20-7.34 (m, 10H), 7.41 (d, 1H, J=8.1), 7.97 (d, 1H,J=7.0), 8.07 (d, 1H, J=7.0); Anal. (C₃₇H₅₃N₅O₈.0.5OH₂O) C, H, N.

Preparation of ProductEthyl-3-{Cbz-L-Leu-L-Phe-L-1-(2-imidazolidone)Ala}-E-Propenoate

TFA (0.8 mL) was added to a solution ofethyl-3-{Cbz-L-Leu-L-Phe-L-(2-Boc-2-aminoethyl)aminoAla}-E-propenoate(0.29 g, 0.42 mmol, 1 equiv.) in CH₂Cl₂ (8 mL), and the reaction mixturewas stirred at 23° C. for 2 h. The volatiles were removed in vacuo, andthe residue was dissolved in EtOAc (25 mL) and washed with saturatedNaHCO₃ solution (25 mL), water (25 mL), and brine (25 mL). The organiclayer was dried over MgSO₄ and was concentrated to give the crudediamine (0.23 g, 92%, 0.39 mmol) as a tan amorphous solid.

This material was dissolved in THF (4 mL), carbonyldiimidazole (0.06 g,0.36 mmol, 0.92 equiv.) was added, and the reaction mixture was stirredat 23° C. for 3.5 h. The solvent was removed in vacuo, and the residuewas purified by flash column chromatography (gradient elution, 0→2%CH₃OH in CH₂Cl₂) to giveethyl-3-{Cbz-L-Leu-L-Phe-L-1-(2-imidazolidone)Ala}-E-propenoate (0.12 g,54%) as a white amorphous solid: mp=161-164° C.; R_(f)=0.21 (5% CH₃OH inCHCl₃); IR (cm⁻¹) 1701, 1647, 1535, 1277; ¹H NMR (DMSO-d₆) δ 0.79 (d,3H, J=6.6), 0.82 (d, 3H, J=6.6), 1.21 (t, 3H, J=7.0), 1.27-1.35 (m, 2H),1.48-1.52 (m, 1H), 2.79-2.86 (m, 1H), 2.92-3.05 (m, 3H), 3.14-3.19 (m,2H), 3.25-3.30 (m, 2H), 3.98-4.03 (m, 1H), 4.10 (q, 2H, J=7.0),4.47-4.49 (m, 1H), 4.59-4.63 (m, 1H), 4.97-5.02 (m, 2H), 5.72 (d, 1H,J=15.8), 6.37 (s, 1H), 6.71 (dd, 1H, J=15.8, 5.5), 7.15-7.39 (m, 10H),7.42 (d, 1H, J=8.1), 8.00 (d, 1H, J=8.1), 8.18 (d, 1H, J=8.1); Anal.(C₃₃H₄₃N₅O₇) C, H, N.

Example 38 Synthesis of Intermediates Q1, Q2, and Q3

Preparation of Intermediate1-Acetyl-3-(triphenylphosphanylidine)-pyrrolidin-2-one

2,4-Dibromobutyride (prepared according to Ikuta et al., J. Med. Chem,1987, vol. 30, 1995) (46.1 g, 188.2 mmol) in THF (1 L) was cooled to 0°C. and treated with a solution of lithium bis(trimethylsilylamide) (40.9g, 244.6 mmol) in THF (200 mL). The solution was held at 0° C. for 2.5h, and then poured into brine (800 mL), extracted with ethyl acetate (2L), and dried (MgSO₄). Evaporation yielded 25.5 g of3-bromo-pyrrolidin-2-one as a brown oil. This material was treated withAc₂O (76 mL) and refluxed 5 hours. Evaporation followed by purification(silica gel filtration, EtOAc elutant) yielded 28 g of1-acetyl-3-bromo-pyrrolidin-2-one as a dark oil. THF (272 mL) andtriphenylphosphine (42.8 g, 163.3 mmol) were added and the resultingsolution was refluxed for 8 hours. Upon cooling to 23° C., a precipitateof 1-acetyl-3-(triphenylphosphanyl)-pyrrolidin-2-one bromide formed andwas collected by filtration (27.1 g). Concentration of the motherliquor, followed by cooling to 0° C., yielded an additional 6.6 g. Thecombined material in CH₂Cl₂ (1 L) was washed with 1N NaOH (100 mL) andthen brine (2×100 mL). Evaporation of the organic layer yielded 26.9 g(37% overall) of 1-acetyl-3-(triphenylphosphanylidine)-pyrrolidin-2-oneas a tan oil. ¹H NMR (CDCl₃) δ 7.76-7.32 (15H, m), 3.90-3.85 (2H, m),2.50 (3H, s), 2.56-2.30 (2H, m).

Preparation of Intermediate2-t-Butoxycarbonyl-3-(t-butyldimethylsilanoxy)-propionic acid methylester

Boc-D-serine methyl ester (20.0 g, 91.2 mmol) in DMF (300 mL) wastreated with imidazole (18.6 g, 273.7 mmol) and then TBSCl (13.0 g, 86.7mmol). The solution was held at room temperature (rt) for 8 h, and thenwashed with saturated aqueous ammonium chloride (300 mL), and extractedwith ethyl acetate (800 mL). The organic layer was washed with brine(300 mL) and then dried (MgSO₄), to yield 30.2 g (100%) of2-t-butoxycarbonyl-3-(t-butyldimethylsilanoxy)-propionic acid methylester as a colorless oil. ¹H NMR (CDCl₃) δ 5.32 (1H, d, J=8.3), 4.33(1H, dt, J=8.8, 2.7), 4.02 (1H, dd, J=10.1, 2.6), 3.80 (1H, dd, J=9.8,3.1), 3.72 (3H, s), 1.43 (9H, s), 0.85 (9H, s), 0.0 (6H, s).

Preparation of Intermediate{1-(1-Acetyl-2-oxo-pyrrolidin-3-ylidenemethyl)-2-(t-butyldimethylsilanyloxy)-ethyl}-carbamicacid t-butyl ester

2-t-Butoxycarbonyl-3-(t-butyldimethylsilanoxy)-propionic acid methylester (12.7 g, 38.0 mmol) in toluene (190 mL) was cooled to −78° C. andtreated with a solution of diisobutylaluminum hydride (15.6 mL, 87.4mmol) in toluene (175 mL). The internal temperature was kept below −70°C. The solution was held at −78° C. for an additional 90 min., and thenmethanol (7.7 mL, 190 mmol) was added.1-Acetyl-3-(triphenylphosphanylidine)-pyrrolidin-2-one (11.1 g, 28.6mmol) in CH₂Cl₂ (50 mL) was added at −78° C., and the resulting solutionwas allowed to warm to room temperature and held for 30 minutes. Asolution of sodium potassium tartrate (150 g) in water (600 mL) wasadded, and stirred vigorously for 30 minutes. The mixture was extractedwith ethyl acetate (4×250 mL), dried (MgSO₄), and evaporated.Purification by silica gel chromatography yielded 7.04 g (60%) of{1-(1-acetyl-2-oxo-pyrrolidin-3-ylidenemethyl)-2-(t-butyldimethylsilanyloxy)-ethyl}-carbamicacid t-butyl ester as a colorless oil. ¹H NMR (CDCl₃) δ 6.59 (1H, dt,J=8.7, 2.9), 4.98 (1H, d, J=6.8), 4.37-4.25 (1H, m), 3.77 (2H, t,J=7.3), 3.70-3.58 (2H, m), 2.90-2.80 (1H, m), 2.75-2.60 (1H, m), 5.53(3H, s), 1.41 (9H, s), 0.87 (9H, s), 0.04 (6H, s).

Preparation of Intermediate{2-Hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acidt-butyl ester

{1-(1-Acetyl-2-oxo-pyrrolidin-3-ylidenemethyl)-2-(t-butyldimethylsilanyloxy)-ethyl}-carbamicacid t-butyl ester (9.18 g, 22.2 mmol) in TBF (150 mL) was treated withTBAF (22.2 mL of a 1M solution in THF, 22.2 mmol) at 0° C., and held for1 hour. A solution of saturated aqueous ammonium chloride was added andstirred for 10 min., and then the solution was extracted with ethylacetate (2×200 mL). The organic layer was dried (MgSO₄) and thenevaporated. Purification by silica gel chromatography yielded 4.82 g(73%) of a colorless oil. This material was taken up in methanol (160mL), treated with potassium carbonate (223 mg, 1.62 mmol), and held for1 h at rt. The mixture was then treated with solid citric acid (311 mg,1.62 mmol), and ethyl acetate (800 mL) was added. The solution wasfiltered through silica gel. Evaporation yielded 4.30 g (73% overall) of{2-hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acidt-butyl ester as a colorless oil. ¹H NMR (CDCl₃) δ 7.03 (1H, br s), 6.35(1H, dt, J=8.6, 2.6), 5.37 (1H, d, J=6.5), 4.40-4.20 (1H, m), 3.66 (brs, 3H), 3.4 (2H, t, J=6.7), 3.10-2.80 (1H, m), 2.80-2.70 (1H, m), 1.41(9H, s).

Preparation of Intermediate{2-Hydroxy-1-(2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acid t-butylester (mixture of diastereomers) (Q1)

{2-Hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acidt-butyl ester (4.30 g, 16.8 mmol) in ethyl acetate (168 mL) was treatedwith 5% palladium on carbon (1.78 g), and hydrogenated at ambientpressure for 1 h. The mixture was filtered and then evaporated to yield3.92 g (91%) of{2-hydroxy-1-(2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acid t-butylester as a colorless oil (1.5:1 mixture of diastereomers): ¹H NMR(CDCl₃) δ 6.99 (1H, s), 5.49 (1H, d, J=8.4), 3.70-3.50 (3H, m),3.38-3.20 (2H, m), 2.60-2.20 (2H, m), 2.00-1.70 (2H, m), 1.65-1.45 (1H,m), 1.37 (9H, s).

Preparation of Intermediate{2-Hydroxy-1-(R-2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acidt-butyl ester (Q2)

{2-Hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acidt-butyl ester (98 mg, 0.39 mmol) in methanol (5 mL) was treated with(R)-BINAP-RuCl (19 mg, 0.02 mmol), and then put under a hydrogenatmosphere (1200 psi) at 50° C. for 48 h. The solution was evaporatedand then filtered through silica gel (10% MeOH-EtOAc elutant).Evaporation yielded 75 mg (75%) of{2-hydroxy-1-(R-2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acidt-butyl ester as a colorless oil (9:1 mixture of diastereomers): ¹H NMR(CDCl₃) δ 6.32 (1H, br s), 5.40(1H, d, J=7.5), 3.82 (1H, br s),3.71-3.63 (2H, m), 3.34-3.31 (2H, m), 2.45-2.30 (2H, m), 2.09-1.86 (2H,m), 1.70-1.63 (1H, m), 1.42 (9H, s).

Preparation of Intermediate{2-Hydroxy-1-(S-2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acidt-butyl ester (Q3)

{2-Hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acidt-butyl ester (0.10 g, 0.39 mmol) in methanol (5 mL) was treated with(S)-BINAP-RuCl (19 mg, 0.02 mmol), and then put under a hydrogenatmosphere (1200 psi) at 50° C. for 48 h. The solution was evaporatedand then filtered through silica gel (10% MeOH-EtOAc elutant).Evaporation yielded 74 mg (74%) of{2-hydroxy-1-(S-2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acidt-butyl ester as a colorless oil (9:1 mixture of diastereomers): ¹H NMR(CDCl₃) δ 6.66 (1H, br s), 5.51 (1H, d, J=8.2), 3.72-3.57 (3H, m),3.34-3.31 (2H, m), 2.52-2.33 (2H, m), 2.20-1.86 (1H, m), 1.86-1.70 (1H,m), 1.62-1.50 (1H, m), 1.40 (9H, s).

Results of biochemical and biological tests conducted using variouscompounds of the invention are described below.

BIOCHEMICAL AND BIOLOGICAL EVALUATION

Inhibition of Rhinovirus 3C Protease

Stock solutions (50 mM, in DMSO) of various compounds were prepared;dilutions were in the same solvent. Recombinant rhinovirus 3C proteases(see Birch et al., “Purification of recombinant human rhinovirus 14 3Cprotease expressed in Escherichia coli,” Protein Expr. Pur. 1995, 6(5),609-618) from serotypes 14, 16, and 2 were prepared by the followingstandard chromatographic procedures: (1) ion exchange using Q SepharoseFast Flow from Pharmacia; (2) affinity chromatography using Affi-GelBlue from Biorad; and (3) sizing using Sephadex G-100 from Pharmacia.Each assay sample contained 2% DMSO, 50 mM tris pH 7.6, 1 mM EDTA, atest compound at the indicated concentration, approximately 1 μMsubstrate, and 50-100 nM protease. The k_(obs)/I values were obtainedfrom reactions initiated by addition of enzyme rather than substrate.RVP activity was measured in the fluorescence resonance energy transferassay. The substrate was (N-terminal)DABCYL-(Gly-Arg-Ala-Val-Phe-Gln-Gly-Pro-Val-Gly)-EDANS (SEQ ID NO. 1).In the uncleaved peptide, the EDANS fluorescence was quenched by theproximal DABCYL moiety. When the peptide was cleaved, the quenching wasrelieved, and activity was measured as an increase in fluorescencesignal. Data were analyzed using standard non-linear fitting programs(Enzfit), and are shown in Tables 1 and 2 below. In Table 1, all dataare for rhinovirus 3C protease from HRV serotype-14 (produced from theinfectious cDNA clone constructed by Dr. Robert Rueckert, Institute forMolecular Virology, University of Wisconsin, Madison, Wis.). Table 2shows protease-inhibiting activity of compounds against proteases fromseveral rhinovirus of serotype other than RVP serotype-14. The data inthe column designated k_(obs)/[I] were measured from progress curves inenzyme start experiments.

Antirhinoviral H1-HeLa Cell Culture Assay

In this cell protection assay, the ability of compounds to protect cellsagainst HRV infection was measured by the XTT dye reduction method,which is described in Weislow et al., J. Natl. Cancer Inst. 1989, vol.81, 577-586.

H1-HeLa cells were infected with HRV-14 at a multiplicity of infection(m.o.i.) of 0.13 (virus particles/cell) or mock-infected with mediumonly. Infected or mock-infected cells were resuspended at 8×10⁵ cellsper mL, and incubated with appropriate concentrations of the compoundsto be tested. Two days later, XTT/PMS was added to test plates and theamount of formazan produced was quantified spectrophotometrically at450/650 nm. The EC₅₀ value was calculated as the concentration ofcompound that increased the percentage of formazan production incompound-treated, virus-infected cells to 50% of that produced bycompound-free, mock-infected cells. The 50% cytotoxic dose (CC₅₀) wascalculated as the concentration of compound that decreased thepercentage of formazan produced in compound-treated, mock-infected cellsto 50% of that produced by compound-free, mock-infected cells. Thetherapeutic index (TI) was calculated by dividing the CC₅₀ value by theEC₅₀ value.

All strains of human rhinovirus (HRV) for use in this assay werepurchased from American Type Culture Collection (ATCC), except for HRVserotype-14 (produced from the infectious cDNA clone constructed by Dr.Robert Rueckert, Institute for Molecular Virology, University ofWisconsin, Madison, Wis.). HRV stocks were propagated and viral assayswere performed in H1-HeLa cells (ATCC). Cells were grown in minimalessential medium with 10% fetal bovine serum, available from LifeTechnologies (Gaithersburg, Md.).

The compounds were tested against control compounds WIN 51711, WIN52084, and WIN 54954 (obtained from Sterling-Winthrop Pharmaceuticals),Pirodavir (obtained from Janssen Pharmaceuticals), and Pleconaril(prepared according to the method described in Diana et al., J. Med.Chem 1995, vol. 38, 1355). Antiviral data obtained for the testcompounds are shown in Tables 1 and 3, where all data are for HRVserotype-14 unless otherwise noted in parentheses. The designation “ND”indicates that a value was not determined for that compound.

TABLE 1 Activity vs. HRV Serotype-14 Protease Cell Inhibition Protectionk_(obs)/[I] EC ₅₀ Compound (M⁻¹sec⁻¹) (μM) Example 31 (ComparisonCompound #2) 25,000 0.61 Example 35 (A-26) 239,000 0.03 Example 33(A-24) 257,000 0.10 Example 36 (A-27) 18,000 1.6 Example 32 (ComparisonCompound #3) 62,500 0.38 Example 34 (A-25) 500,000 0.03 Example 5 (A-4)270,000 0.10 Example 8 (A-7) 980,000 0.004 Example 7 (A-6) 248,000 0.42Example 9 (A-8) 900,000 ND Example 6 (A-5) 1,500,000 0.005 Example 12(C-1) 68,400 0.10 Example 18 (C-2) 270,000 0.002 Example 10 (B-1)240,000 0.10 Example 20 (B-4) 500,000 <0.03 Example 17 (B-2) 1,090,0000.005 Example 1 (Comparison Compound #1) 573 >320 Example 2 (A-1)260,000 0.25 Example 3 (A-2) 46,900 1.6 Example 4 (A-3) 310,000 0.05Example 11 (A-10) 108,000 0.14 Example 13 (A-11) 108,000 0.03 Example 14(A-9) 66,000 1.8 Example 15 (A-12) 59,300 0.40 Example 16 (A-13) 95,8000.20 Example 19 (B-3) 465,000 0.18 Example 21 (A-14) 54,500 0.48 Example22 (A-15) 237,100 0.22 Example 23 (A-16) 172,800 0.45 Example 24 (A-17)167,000 0.06 Example 25 (A-18) 292,000 1.5 Example 26 (A-19) 27,750 25.1Example 27 (A-20) 1,020 12.6 Example 28 (A-21) 17,800 2.5 Example 29(A-22) 2,400 nd Example 30 (A-23) 26,000 nd

TABLE 2 Activity vs. Other HRV Serotypes Compound Rhinovirus Serotypek_(obs/I) (M⁻¹sec⁻¹) Comparison Compound #2 (16) 6,500 ″ (89) 3,400 ″ (2) 2,000 Comparison Compound #3  (2) 8,000 ″ (16) 16,900  (A-24)  (2)31,000 

TABLE 3 Anti-Rhinovirus Activity # HRV EC₅₀ (μM) CC₅₀ (μM) TIComparison >320 >320 — Compound #1 (A-1) 0.25 >100 >400 ″  (2) 0.41″ >243 ″ (1A) 1.0 ″ >100 ″ (89) 0.22 ″ >450 (A-2) 1.6 >100 >63 (A-3)0.05   >10 >200 (A-4) 0.10 >100 >1000 (A-5) 0.005  >10 >2000 ″  (2) 0.01″ >1000 ″ (16) 0.02 ″ >500 ″ (39) 0.02 ″ >500 ″ (89) 0.02 ″ >500 ″ (10)0.05 ″ >200 ″ (1A) 0.03 ″ >333 ″  (3) 0.05 ″ >200 ″  (9) 0.04 ″ >250 ″(12) 0.06 ″ >166 ″ (13) 0.02 ″ >500 ″ (17) 0.02 ″ >500 ″ (25) 0.18 ″ >55″ (30) 0.06 ″ >166 ″ (38) 0.13 ″ >76 ″ (87) 0.21 ″ >47 (A-6)0.42 >100 >237 (A-7) 0.004   >10 >2500 ″  (2) 0.02 ″ >500 ″ (16) 0.04″ >250 ″ (39) 0.02 ″ >500 ″ (89) 0.02 ″ >500 ″ (10) 0.06 ″ >166 ″ (1A)0.03 ″ >333 ″  (3) 0.02 ″ >500 ″  (9) 0.04 ″ >250 ″ (12) 0.07 ″ >143 ″(13) 0.03 ″ >333 ″ (17) 0.02 ″ >500 ″ (25) 0.20 ″ >50 ″ (30) 0.09 ″ >111″ (38) 0.17 ″ >58 ″ (87) 0.59 ″ >16 (A-8) ND ND ND (B-1) 0.10 >100 >1000″ (1A) 0.30 ″ >333 ″ (10) 0.40 ″ >250  (A-10) 0.14 >100 >714 (C-1) 0.10 >10 >100  (A-11) 0.03   50 1667 (A-9) 1.8 >100 >55  (A-12)0.40 >100 >250  (A-13) 0.20  >10 >50 (B-2) 0.005 >100 >20000 ″  (2) 0.02″ >5000 ″ (16) 0.01 ″ >10000 ″ (39) 0.05 ″ >2000 ″ (89) 0.009 ″ >11111 ″(10) 0.02 ″ >5000 ″ (1A) 0.02 ″ >5000 ″  (3) 0.02 ″ >5000 ″  (9) 0.006″ >16666 ″ (12) 0.05 ″ >2000 ″ (13) 0.01 ″ >10000 ″ (17) 0.01 ″ >10000 ″(25) 0.03 ″ >3333 ″ (30) 0.04 ″ >2500 ″ (38) 0.07 ″ >1428 ″ (87) 0.06″ >1666 (C-2) 0.002   >10 >5000 ″  (2) 0.004 ″ >2500 ″ (16) 0.01 ″ >1000″ (39) 0.01 ″ >1000 ″ (89) 0.004 ″ >2500 ″ (10) 0.02 ″ >500 ″ (1A) 0.01″ >1000 ″  (3) 0.02 ″ >500 ″  (9) 0.01 ″ >1000 ″ (12) 0.04 ″ >250 ″ (13)0.007 ″ >1428 ″ (17) 0.007 ″ >1428 ″ (25) 0.07 ″ >142 ″ (30) 0.03 ″ >333″ (38) 0.05 ″ >200 ″ (87) 0.02 ″ >500 (B-3) 0.18 >100 >543 (B-4)<0.03 >100 >3333  (A-14) 0.48 >100 >208  (A-15) 0.22 >100 >454 ″ (1A)7.1 ″ >14 ″ (10) 2.7 ″ >37   (A-16) 0.45 >100 >222 ″ (1A) 4.8 ″ >21 ″(10) 4.5 ″ >22   (A-17) 0.06 >100 >1786 ″ (1A) 1.8 ″ >56 ″ (10) 3.3″ >30  (A-18) 1.5 >100 >67  (A-19) 25.1 >100 >4  (A-20) 12.6 >100 >8 (A-21) 2.5 >100 >40  (A-22) ND ND ND  (A-23) ND ND ND Comparison0.61 >320 >524 Compound #2 Comparison (16) 2.3 >320 >139 Compound #2Comparison (89) 6.3 >320 >50 Compound #2 Comparison (10) 0.60 >320 >533Compound #2 Comparison 0.38 >320 >842 Compound #3   (A-24)0.10 >100 >1000   (A-25) 0.030 >100 >3333   (A-26) 0.030 >100 >3333  (A-27) 1.6 >100 >62 WIN 51711 0.78  >60 >77 WIN 52084 0.07  >10 >143 WIN54954 2.13  >63 >30 Pirodavir 0.03  >10 >300 Pleconaril 0.11  >10 >1000

Anticoxsackieviral Cell Culture Assay

Coxsackievirus types A-21 (CAV-21) and B3 (CVB-3) were purchased fromAmerican Type Culture Collection (ATCC, Rockville, Md.). Virus stockswere propagated and antiviral assays were performed in H1-HeLa cells(ATCC). Cells were grown in minimal essential medium with 10% fetalbovine serum (Life Technologies, Gaithersburg, Md.).

The ability of compounds to protect cells against either CAV-21 or CVB-3infection was measured by the XTT dye reduction method. This method isdescribed in Weislow et al., J. Natl. Cancer Inst. 1989, vol. 81,577-586. H1-HeLa cells were infected with CAV-21 or CVB-3 at amultiplicity of infection (m.o.i.) of 0.025 or 0.075, respectively, ormock-infected with medium only. H1-HeLa cells were plated at 4×10⁴ cellsper well in a 96-well plate and incubated with appropriateconcentrations of the test compound. One day (CVB-3) or two days(CAV-21) later, XTT/PMS was added to test plates and the amount offormazan produced was quantified spectrophotometrically at 450/650 nm.The EC₅₀ was calculated as the concentration of compound that increasedthe formazan production in compound-treated, virus-infected cells to 50%of that produced by compound-free, uninfected cells. The 50% cytotoxicdose (CC₅₀) was calculated as the concentration of compound thatdecreased formazan production in compound-treated, uninfected cells to50% of that produced in compound-free, uninfected cells. The therapeuticindex (TI) was calculated by dividing the CC₅₀ by the EC₅₀.

The compounds were tested against control compounds WIN 54954 (obtainedfrom Sterling-Winthrop Pharmaceuticals), Pirodavir (obtained fromJanssen Pharmaceuticals), and Pleconaril (prepared according to Diana etal., J. Med. Chem. 1995, 38, 1355). Antiviral data obtained for the testcompounds against CAV-21 and CVB-3 are shown in Table 4.

TABLE 4 Anti-Coxsackievirus Activity Compound Strain EC₅₀ (μM) CC₅₀ (μM)TI (A-5) CAV-21 0.23  >10 >43 ″ CVB-3 1.0 ″ >10 (B-2) CAV-210.16 >100 >625 ″ CVB-3 0.18 ″ >555 WIN 54954 CAV-21 >100 >100 ″CVB-3 >100 ″ Pirodavir CAV-21 >100 >100 ″ CVB-3 >100 ″ Pleconaril CAV-210.09  >10 >107 ″ CVB-3 >10 ″

Anti-Echoviral and -Enteroviral Cell Culture Assays

Echovirus type 11 (EV 11) and enterovirus type 70 (EV 70) were purchasedfrom ATCC (Rockville, Md.). Virus stocks were propagated and antiviralassays were performed in MRC-5 cells (ATCC). Cells were grown in minimalessential medium with 10% fetal bovine serum (Life Technologies,Gaithersburg, Md.).

The ability of compounds to protect cells against either EV 11 or EV 70infection was measured by the XTT dye reduction method (Weislow et al.,J. Natl. Cancer Inst. 1989, vol. 81, 577-586). MRC-5 cells were infectedwith EV 11 or EV 70 at an m.o.i. of 0.003 or 0.004, respectively, ormock-infected with medium only. Infected or uninfected cells were addedat 1×10⁴ cells per well and incubated with appropriate concentrations ofcompound. Four days later, XTT/PMS was added to test plates, and theamount of formazan produced was quantified spectrophotometrically at450/650 nm. The EC₅₀ was calculated as the concentration of compoundthat increased the formazan production in compound-treated,virus-infected cells to 50% of that produced by compound-free,uninfected cells. The 50% cytotoxic dose (CC₅₀) was calculated as theconcentration of compound that decreased formazan production incompound-treated, uninfected cells to 50% of that produced incompound-free, uninfected cells. The therapeutic index (TI) wascalculated by dividing the CC₅₀ by the EC₅₀.

The compounds were tested against control compounds Pirodavir (obtainedfrom Janssen Pharmaceuticals) and Pleconaril (prepared according toDiana et al., J. Med. Chem. 1995, vol. 38, 1355). Antiviral dataobtained for the test compounds against strain EV 11 and EV 70 are shownbelow in Table 5.

TABLE 5 Anti-Echovirus and Anti-Enterovirus Activity Compound StrainEC₅₀ (μM) CC₅₀ (μM) TI (A-5) EV-11 0.08 >10 >125 ″ EV-70 0.04 ″ >250(B-2) EV-11 0.01 >100  >10000 ″ EV-70 0.003 ″ >33333 Pirodavir EV-113.7 >10 >3 ″ EV-70 0.06 ″ >167 Pleconaril EV-11 0.16 >10 >62 ″ EV-70 NDND ND

While the invention has been described in terms of preferred embodimentsand specific examples, those skilled in the art will recognize thatvarious changes and modifications can be made without departing from thespirit and scope of the invention. Thus, the invention should beunderstood as not being limited by the foregoing detailed description,but as being defined by the appended claims and their equivalents.

1 1 10 PRT Artificial Sequence A forced amino acid consensus sequence ofthe rhinovirus 3C protease cleavage site wherein certain amino acidshave been substituted. 1 Gly Arg Ala Val Phe Gln Gly Pro Val Gly 1 5 10

What is claimed is:
 1. A compound of the formula I:

wherein: Y is —C(R_(y))(R_(y))— where each R_(y) is independently H orlower alkyl; R₁ is H, F, an alkyl group, OH, SH, or an O-alkyl group; R₂and R₃ are each independently H;

 where n is an integer from 0 to 5, A₁ is CH or N, A₂ and each A₃ areindependently selected from C(R₄₁)(R₄₁), N(R₄₁), S, S(O), S(O)₂, and O,and A₄ is NH or NR₄₁, where each R₄₁ is independently H or lower alkyl,provided that no more than 2 heteroatoms occur consecutively in the ringformed by A₁, A₂, (A₃)_(n), A₄ and C═O; and provided that at least oneof R₂ and R₃ is

R₅ and R₆ are each independently H, F, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group or a heteroaryl group; R₇and R₈ are each independently H, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, or an aryl group, or a heteroaryl group, —OR₁₇,—SR₁₇, —NR₁₇R₁₈, —NR₁₉NR₁₇R₁₈, or —NR₁₇OR₁₈, where R₁₇, R₁₈, and R₁₉ areeach independently H, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an aryl group, a heteroaryl group, or an acylgroup; R₉ is a five-membered heterocycle having from one to threeheteroatoms selected from O, N, and S, or R₉ is

Z and Z₁ are each independently H, F, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, a heteroaryl group,—C(O)R₂₁, —CO₂R₂₁, —CN, —C(O)NR₂₁R₂₂, —C(O)NR₂₁OR₂₂, —C(S)R₂₁,—C(S)NR₂₁R₂₂, —NO₂, —SOR₂₁, —SO₂R₂₁, —SO₂NR₂₁R₂₂, —SO(NR₂₁)(OR₂₂),—SONR₂₁, —SO₃R₂₁, —PO(OR₂₁)₂, —PO(R₂₁)(R₂₂), —PO(NR₂₁R₂₂)(OR₂₃),PO(NR₂₁R₂₂)(NR₂₃R₂₄), —C(O)NR₂₁NR₂₂R₂₃, or —C(S)NR₂₁NR₂₂R₂₃, where R₂₁,R₂₂, R₂₃, and R₂₄ are each independently H, an alkyl group, a cycloalkylgroup, a heterocycloalkyl group, an aryl group, a heteroaryl group, anacyl group, or a thioacyl group, or any two of R₂₁, R₂₂, R₂₃, and R₂₄,together with the atom(s) to which they are bonded, form aheterocycloalkyl group, provided that Z and Z₁ are not both H; or Z₁ andR₁, together with the atoms to which they are bonded, form a cycloalkylor heterocycloalkyl group; or Z and Z₁, together with the atoms to whichthey are bonded, form a cycloalkyl or heterocycloalkyl group; or solvatethereof.
 2. A compound or solvate according to claim 1, wherein R₂ andR₃ are each independently H;

where n is an integer from 0 to 5, each R₄₁ is independently H or loweralkyl, and the stereochemistry at the carbon denoted with an asterisk(*) may be R or S; provided that at least one of R₂ and R₃ is


3. A compound or solvate according to claim 1, wherein Y is —N(R_(y))—,where R_(y) is H or lower alkyl.
 4. A compound or solvate according toclaim 3, wherein: Z and Z₁ are each independently selected from H, F,lower alkyl, —CO₂R₂₁, and —C(O)NR₂₁R₂₂, where R₂₁ and R₂₂ are eachindependently H, an alkyl group, a cycloalkyl group, a heterocycloalkylgroup, an aryl group, a heteroaryl group, an acyl group, or a thioacylgroup, or R₂₁ and R₂₂, together with the atom(s) to which they arebonded, form a heterocycloalkyl group, provided that Z and Z₁ are notboth H; or Z₁ and R₁, together with the atoms to which they are bonded,form a cycloalkyl or heterocycloalkyl group; or Z and Z₁, together withthe atoms to which they are bonded, form a cycloalkyl orheterocycloalkyl group.
 5. A compound or solvate according to claim 3,wherein R₁ is H, F, or methyl.
 6. A compound or solvate according toclaim 3, wherein at least one of R₂ or R₃ is


7. A compound or solvate according to claim 6, wherein one of R₅ and R₆is H and the other is alkyl or aryl.
 8. A compound or solvate accordingto claim 3, wherein one of R₅ and R₆ is H and the other is alkyl oraryl.
 9. A compound or solvate according to claim 3, wherein one of R₅and R₆ is H and the other is unsubstituted or substituted phenylmethyl.10. A compound or solvate according to claim 3, wherein R₇ and R₈ areeach independently H, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an aryl group, or a heteroaryl group.
 11. Acompound or solvate according to claim 3, wherein one of R₇ and R₈ is Hand the other is alkyl or aryl.
 12. A compound or solvate according toclaim 3, wherein one of R₇ and R₈ is H and the other is 2-propyl,2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl, or naphthylmethyl.13. A compound or solvate according to claim 3, wherein R₉ is afive-membered heterocycle having at least one nitrogen heteroatom andone oxygen heteroatom.
 14. A compound or solvate according to claim 3,wherein R₉ is selected from substituted and unsubstituted 1,2-oxazolyl,1,3-oxazolyl, and 1,2,4-oxadiazolyl.
 15. A compound or solvate accordingto claim 3, wherein R₉ is 3-isoxazolyl or 5-isoxazolyl unsubstituted orsubstituted with one or two substituents selected from methyl andhalogens.
 16. A method of inhibiting picornavirus replication in amammal afflicted with meningitis or hepatitis A comprising administeringa compound according to claim 1 to a mammal for a time and underconditions effective to inhibit picornavirus replication.
 17. A compoundaccording to claim 1 of the formula:

or a solvate thereof.
 18. A compound which is a metabolite of a compoundaccording to claim 1, wherein said metabolite is effective to inhibitreplication of a picornavirus.
 19. A composition comprising anacceptable carrier and a compound according to claim 1 in an amounteffective to inhibit replication of a picornavirus.
 20. A compound orsolvate according to claim 1, wherein Y is —CH₂—.
 21. A compound orsolvate according to claim 20, wherein one of R₅ and R₆ is H and theother is alkyl or aryl.
 22. A compound or solvate according to claim 20,wherein one of R₅ and R₆ is H and the other is unsubstituted orsubstituted phenylmethyl.
 23. A compound or solvate according to claim20, wherein R₇ and R₈ are each independently H, an alkyl group, acycloalkyl group, a heterocycloalkyl group, an aryl group, or aheteroaryl group.
 24. A compound or solvate according to claim 20,wherein one of R₇ and R₈ is H and the other is alkyl or aryl.
 25. Acompound or solvate according to claim 20, wherein one of R₇ and R₈ is Hand the other is 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl,phenylmethyl, or naphthylmethyl.
 26. A compound or solvate according toclaim 20, wherein R₉ is a five-membered heterocycle having at least onenitrogen heteroatom and one oxygen heteroatom.
 27. A compound or solvateaccording to claim 26, wherein R₉ is unsubstituted or substituted1,2-oxazolyl, 1,3-oxazolyl, or 1,2,4-oxadiazolyl.
 28. A compound orsolvate according to claim 27, wherein R₉ is 3-isoxazolyl or5-isoxazolyl unsubstituted or substituted with one or two substituentsselected from methyl and halogens.
 29. A compound or solvate accordingto claim 20, wherein at least one of R₂ and R₃ is


30. A compound or solvate according to claim 29, wherein one of R₅ andR₆ is H and the other is alkyl or aryl.
 31. A compound or solvateaccording to claim 20, wherein R₁ is H, F, or methyl.
 32. A compound orsolvate according to claim 20, wherein Z and Z₁ are each independentlyselected from H, F, lower alkyl, —CO₂R₂₁, and —C(O)NR₂₁R₂₂, where R₂₁and R₂₂ are each independently H, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an aryl group, a heteroaryl group, an acylgroup, or a thioacyl group, or R₂₁ and R₂₂, together with the atom(s) towhich they are bonded, form a heterocycloalkyl group, provided that Zand Z₁ are not both H; or Z₁ and R₁, together with the atoms to whichthey are bonded, form a cycloalkyl or heterocycloalkyl group; or Z andZ₁, together with the atoms to which they are bonded, form a cycloalkylor heterocycloalkyl group.
 33. A compound according to claim 20 of theformula I-B″:

wherein R₁, R₂, R₆, R₇, R₉, Z, and Z₁ are as defined in claim 20, or asolvate thereof.
 34. A compound or solvate according to claim 33,wherein R₁ is H and: R₂ is CH₂CH₂C(O)NH₂, R₆ is

 R₇ is CH(CH₃)₂, Z is H, Z₁ is CO₂CH₂CH₃, and R₉ is

R₂ is

 R₇ is CH(CH₃)₂, Z is H, Z¹ is CO₂CH₂CH₃, and R₉ is

 R₇ is CH(CH₃)₂, Z and Z₁ together are

 where the carbonyl group is cis to the R₁ hydrogen, and  R₉ is

 or

 R₇ is CH(CH₃)₂, Z is H, Z₁ is CO₂CH₂CH₃, and R₉ is


35. A compound or solvate according to claim 33, wherein R₂ is


36. A compound or solvate according to claim 33, wherein: R₁ is H, F, ormethyl; Z and Z₁ are each independently selected from H, F, —CO₂R₂₁,—CN, and —C(O)NR_(21,)R₂₂, where R₂₁ and R₂₂ are each independently H,an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an arylgroup, a heteroaryl group, an acyl group, or a thioacyl group, or R₂₁and R₂₂, together with the atom(s) to which they are bonded, form aheterocycloalkyl group, provided that Z and Z₁ are not both H; or Z₁ andR₁, together with the atoms to which they are bonded, form a cycloalkylor hererocycloalkyl group; or Z and Z₁, together with the atoms to whichthey are bonded, form a cycloalkyl or heterocycloalkyl group; R₂ isselected from

R₆ is unsubstituted or substituted phenylmethyl; R₇ is alkyl or aryl;and R₉ is 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted withone or two substituents selected from methyl and halogens.
 37. Acompound or solvate according to claim 36, wherein R₇ is selected from2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl, andnaphthylmethyl.
 38. A compound having a formula:

wherein: R₁ is H, F, an alkyl group, OH, SH, or an O-alkyl group; Z andZ₁ are each independently H, F, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an aryl group, a heteroaryl group, —C(O)R₂₁,—CO₂R₂₁, —CN, —C(O)NR₂₁R₂₂, —C(O)NR₂₁OR₂₂, —C(S)R₂₁, —C(S)NR₂₁R₂₂, —NO₂,—SOR₂₁, —SO₂R₂₁, —SO₂NR₂₁R₂₂, —SO(NR₂₁)(OR₂₂), —SONR₂₁, —SO₃R₂₁,—PO(OR₂₁)₂, —PO(R₂₁)(R₂₂), —PO(NR₂₁R₂₂)(OR₂₃), PO(NR₂₁R₂₂)(NR₂₃R₂₄),—C(O)NR₂₁NR₂₂R₂₃, or —C(S)NR₂₁NR₂₂R₂₃, where R₂₁, R₂₂, R₂₃, and R₂₄ areeach independently H, an alkyl group, a cycloalkyl group, aheterocycloalkyl group, an aryl group, a heteroaryl group, an acylgroup, or a thioacyl group, or any two of R₂₁, R₂₂, R₂₃, and R₂₄,together with the atom(s) to which they are bonded, form aheterocycloalkyl group, provided that Z and Z₁ are not both H; or Z₁ andR₁, together with the atoms to which they are bonded, form a cycloalkylor heterocycloalkyl group; or Z and Z₁, together with the atoms to whichthey are bonded, form a cycloalkyl or heterocycloalkyl group; n is 1 or2; R_(y) is H or lower alkyl, R₆ is alkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, arylthiomethyl or heteroaryl; R₇ is alkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, —OR₁₇, —SR₁₇, —NR₁₇R₁₈,—NR₁₉NR₁₇R₁₈, or —NR₁₇OR₁₈, where R₁₇, R₁₈, and R₁₉ are eachindependently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,or acyl; and R₉ is a five-membered heterocycle having one to threeheteroatoms selected from O, N, and S, or R₉ is

 or a solvate thereof.
 39. A compound or solvate according to claim 38,wherein: R₁ is H, F, or alkyl; R_(y) is H or methyl; R₆ is alkyl,arylalkyl or arylthiomethyl; R₇ is alkyl, cycloalkyl, heterocycloalkyl,aryl, or heteroaryl; R₉ is a five-membered heterocycle having from oneto three heteroatoms selected from O, N, and S, where at least one ofthe heteroatoms is nitrogen, that is unsubstituted or substituted withone or two substituents selected from lower alkyl groups and halogens;or a solvate thereof.
 40. A compound or solvate according to claim 38,wherein: R₆ is arylmethyl or arylthiomethyl; R₇ is an alkyl group; R₉ is3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one ortwo substituents selected from methyl and halogens; and Z is H and Z₁ is—CO₂R₂₁, —CN, or —C(O)NR₂₁R₂₂, where R₂₁ and R₂₂ are each independentlyH, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an arylgroup, a heteroaryl group, an acyl group, or a thioacyl group, or R₂₁and R₂₂ together with the atom(s) to which they are bonded form aheterocycloalkyl group, or Z and Z₁ together form a cyclic ester oramide.
 41. A compound or solvate according to claim 40, wherein: R₁ is Hor F; R₆ is arylmethyl or arylthiomethyl; and R₇ is selected from2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, and arylmethyl.
 42. Acompound or solvate according to claim 41, wherein R₆ is phenylmethyl,where the phenyl moiety optionally has one to three substituentsselected from halogen, lower alkyl, and lower alkoxy.
 43. A compoundaccording to claim 38, selected from the group consisting of

or a solvate thereof.
 44. A compound according to claim 38, wherein R₆is arylalkyl or arylthiomethyl and the aryl moiety thereof issubstituted with a halogen, alkyl or alkoxy group.
 45. A compoundaccording to any one of claim 9, 22 or 36, wherein R₆ is substitutedphenylmethyl, wherein the phenyl moiety thereof is substituted with ahalogen.
 46. A method of inhibiting picornavirus replication in a mammalin need thereof comprising administering a compound according to claim 1to a mammal for a time and under conditions effective to inhibitpicornavirus replication.
 47. A method of inhibiting picornavirusreplication in a mammal comprising administering a compound according toclaim 1 to a mammal for a time and under conditions effective to inhibitpicornavirus replication, wherein said mammal is infected with apicornavirus.
 48. A method of inhibiting a picornaviral 3C protease in amammal that is infected with a picornavirus comprising administering tosaid mammal a compound according to claim 1, for a time and undercondition effective to inhibit picornaviral 3C protease.
 49. A method ofinhibiting replication of a picornavirus comprising: contacting apicornavirus with a compound or solvate as defined in claim 1 for a timeand under conditions effective to inhibit picornavirus 3C protease. 50.A method as defined in claim 49, wherein the picornaviral 3C protease isa rhinoviral protease.